Methods and compositions for the use of apurinic/apyrimidinic endonucleases

ABSTRACT

Disclosed are methods and compositions for identifying, monitoring and treating premalignant and malignant conditions in a human subject. The present invention further discloses methods and compositions for determining cells undergoing apoptosis, and for increasing the efficacy of a cancer therapy. The methods involve the use of apurinic/apyrimidinic endonuclease (APE), independently, as a marker for (pre)malignant conditions and for apoptosis. Also described are polyclonal antibody preparations for use in methods for detecting APE and methods for modulating expression susceptibility of cells to apoptosis.

This application in is a continuation-in-part of U.S. Provisional PatentApplication No. 60/019,561, filed Jun. 11, 1996 and U.S. ProvisionalPatent Application No. 60/019,602, filed Jun. 11, 1996. The entire textof each of the above-referenced disclosures is specifically incorporatedby reference herein without disclaimer.

BACKGROUND OF THE INVENTION

A. Field of the Invention

The present invention relates generally to the fields of molecularbiology, gene regulation and pathology. More specifically, in certainaspects, the invention relates to the identification of premalignant ormalignant conditions in tissues. In other aspects, the invention relatesto methods and compositions for the identification of apoptosis, orprogrammed death, in cells. In particular, the present invention relatesto monitoring of levels of apurinic/apyrimidinic endonucleases, alsoknown as APE's.

B. Related Art

(i) Cancer Markers

Despite continued efforts worldwide to identify useful prognosticfactors for premalignant and malignant conditions (hereinafter togetherreferred to as "(pre)malignant conditions") of human tissues, relativelyfew markers and associated screens have been discovered which reliablyidentify (pre)malignant conditions. As one specific example, squamouscell carcinoma of the cervix uteri (SCCC) is the second most commonfemale malignancy, and the leading cause of death by cancer in womenworldwide (Mitchell et al., 1995; Burger et al., 1995; Richard et al.,1995). Based on recent estimates, there will be approximately 15,800 newcases of invasive disease, 65,000 cases of carcinoma in situ (CIS;premalignant), and 4800 deaths attributed to SCCC annually in the UnitedStates alone (American Cancer Society, 1995). African- andHispanic-American women and poor Caucasian women were found to have amortality rate from cervical cancer of more than double that of allCaucasian-American women (Burger et al., 1993; Miller et al., 1993;Davis et al., 1995; Parham et aL 1995), and HIV positive women werediagnosed with CIS at five times the rate of HIV negative women (Wrightet al., 1994; Heard et al., 1995).

Cervical cancer arises in the squamous cells lining the cervix tissue.Precancerous lesions are known as CIS, dysplasia, or cervicalintraepithelial neoplasia (CIN). Although the development of these cellsinto invasive carcinoma can take ten to twelve years, in about 10% ofpatients the development is much more rapid, occurring in less than ayear (National Cancer Institute, 1995). Early detection of cervicalcancer substantially increases the probability of survival, with bothmalignant and premalignant conditions being detectable by the so-calledPap smear.

While the Pap smear is relatively widely used, and has had an overallpositive impact on women's health, it presents several significantdrawbacks. Pap smear sampling must be performed by highly trainedclinicians to result in an interpretable, representative sample of thecells lining the cervix (Koss, 1989). As well, a trained cytologist mustanalyze the morphology of the cells upon microscopic examination (Koss,1989). Significant human error is attributed to both steps, contributingto high levels of false-negative readings (Koss, 1989; Koss, 1993). Amajority of studies estimate the rate of false-negatives at 20%-30%,with various other studies putting this value at 5% to in excess of 50%(Morell et al., 1982). In addition, subsequent to a positiveinterpretation of a Pap smear, a physician typically biopsies thecervical tissue to confirm the diagnosis and assist in the determinationof the stage of the disease and the design of an appropriate treatmentregimen. The biopsy sample is analyzed by a pathologist for the presenceor absence of (pre)malignant cells and to determine the extent of tumorgrowth. Human error can also arise in these procedures (Sideri, et al.,1982).

In light of these and other shortcomings of the common Pap smear,researchers have been actively seeking a reliable marker for(pre)malignant states in cervical tissue. A useful marker and associatedassay are understood to require a number of attributes. An assay usingthe marker must consistently detect differences in cancer and noncancer,and exhibit both specificity (few false positives) and sensitivity (fewfalse negatives). Quantitative assays find increased utility over thosewhich are merely qualitative, and cancer marker specific for aparticular organ or cell type will be more useful for initial screeningpurposes, but organ/cell specificity is less important for monitoringpreviously diagnosed patients.

A number of putative markers for (pre)malignant conditions of the cervixhave been identified; however, the markers suggested to date exhibitseveral shortcomings. For instance, squamous cell carcinoma antigen is aglycoprotein purified from SCCC that has been found to be a marker forcancerous conditions of the cervix (Kato et al., 1982; Kato et al.,1984). This marker was originally called T-4 in a lesser-purified form,and serum SCCA was found to be elevated in 61% of SCCC cases overall,ranging from 30-45% in Stage 1 to 90-100% in Stage 4 (Crombach et al.,1989) (FIGO classification, National Cancer Institute, 1995). Inoriginal testing of SCCC as a tissue marker using flow cytometry ofvaginal smear cells, 85% of SCCC cases, 80% of severe and 43% of mild tomoderate dysplasias and 21% of normal specimens contained cells stainedwith antibodies to SCCA (Suehiro et al., 1986). This lack of specificitydecreases the usefulness of SCCA as a marker, which has also beenbolstered by the observation that cytosolic concentration of SCCA innormal cells is twice as high in normal cells than in SCCC cells(Crombach et al., 1989).

Another putative marker for SCCC is carcinoembryonic antigen (CEA). Oneof the most studied antigens using immunohistochemical analysis for thedetermination of neoplastic cells in SCCC is CEA. Reports as to thepercentage of different dysplastic and neoplastic lesions stained havevaried (Toki et al., 1991; Rutenan et al., 1978; van Nagell et al.,1982; Bamford et al., 1983; Bychkov et al., 1983; McDiken et al., 1983;Lindgren et al, 1986; Agarwal et al., 1990). Additional possible markerswhich have been studied, with varying degrees of success, includeproliferating cell nuclear antigen (PCNA) (Raju, 1994; Steinbeck et al.,1995), epithelial membrane antigen (EMA) (Bamford et al., 1983; Sarkeret al., 1994), various keratins (Rajur et al., 1988; Auger et al.,1990), Tn antigen (Hamada et al., 1993; Hirao et al., 1993), oncogenesand tumor suppressor genes (Kohier et al., 1989; Tervahauta et al.,1994; Hale et al., 1993; Terzano et al., 1993; Sainz et al., 1993;Tervahauta et al., 1993; Cardillo et al., 1993), and various others(Fuchs et al., 1989; Flint et al., 1988; Costa et a., 1987; Lara et al.,1994; Carico et al., 1993; Harlozinski et al., 1985).

(ii) APE

Apurinic/apyrimidinic endonucleases (hereinafter sometimes referred toas "apurinic endonuclease" or "APE") catalyze repair of baseless sitesin DNA. At least 10,000-20,000 of these sites are generated daily inevery human cell as a result of oxidation, spontaneous hydrolysis, andthe removal of modified bases by DNA glycosylases (Loeb, 1985, FIG. 15).These baseless sites disrupt transcription and are highly mutagenic ifnot repaired.

The major human apurinic/apyrinmidinic endonuclease is a 37,000 Daltonprotein which has been cloned and shown to complement APE deficientbacteria (Demple et al., 1991). APE has been shown to be identical toRef-1, a redox factor facilitating the DNA binding of a number oftranscription factors, many of which are important in oncogenesis,including Fos, Jun, Myb, and members of the ATF/CREB family(Xanthoudakis et al., 1992). Recently, APE has also been shown to beinvolved in the negative regulation of transcription of the parathyroidhormone gene by extracellular calcium in vitro (Okazaki et al., 1994).Ape also appears to be a major regulator of p53 activity, acting throughprotein modification of p53 (Jayaraman et al., 1997)

Besides DNA repair activity, the major human APE repair enzyme has beenfound to exhibit multiple flnctions, many by in vitro studies. Forexample, investigators studying Ref-1, a redox regulating transcriptionfactor, discovered that Ref-1 and APE were identical (Xanthoudakis etal., 1992). APE/Ref-1 facilitates the DNA binding characteristics ofJun-Jun homodimers, Fos-Jun heterodimers, HeLa AP-1, and numerous othertranscription factors, including Myb, members of the CREB family andnuclear factor-κB (Xanthoudakis et al., 1992).

Immunohistochemistry has been used to examine the subcellulardistribution of APE in several different human tissues. The results showthat levels vary significantly in different tissues (Duguid et al.,1995). APE expression in skin and intestine was tightly linked tocellular maturation. In most tissues, APE was detected primarily in thenucleus, where the APE staining pattern followed that of chromatin. Inhepatocytes and some neurons, however, APE was detected primarily in thecytoplasm.

At this point in time, APE has not been associated with any particularpathologic conditions. Though clearly important to cellular function,specific diseases resulting from aberrations in this protein's functionare not known.

SUMMARY OF THE INVENTION

The present invention provides a method for identifying a premalignantor malignant condition in a human subject comprising determining thelevel of APE in cells from a sample from the human subject, wherein anelevated level of APE, as compared to the APE level in correspondingnormal cells, indicates a premalignant or malignant condition in thehuman subject. As used herein the term "normal cells" means cells of thesame tissue type, grown and at the same conditions and at the same cellcycle window and state of differentiation. In particular embodiments,the sample is selected from the group consisting of skin, muscle, facia,brain, prostate, breast, endometrium, lung, pancreas, small intestine,blood cells, liver, testes, ovaries, cervix, colon, skin, stomach,esophagus, spleen, lymph node, bone marrow, kidney, peripheral blood,lymph fluid, ascites, serous fluid, pleural effusion, sputum,cerebrospinal fluid, lacrimal fluid, stool and urine.

In other embodiments, the premalignancy or malignancy is selected fromthe group consisting of brain, lung, liver, spleen, kidney, lymph node,small intestine, pancreas, blood cells, colon, stomach, breast,endometrium, cervix, prostate, testicle, ovary, skin, head and neck,esophagus, bone marrow and blood tumor cells.

In a particular aspect the determining comprises evaluating APE proteinlevels. In some aspects, the determining may comprise evaluating APEtranscript levels. In other aspects, the evaluating may comprise aninununoassay. In those aspects where APE transcript levels are evaluatedthe present invention may employ quantitative RT-PCR.

The present invention also provides an polyclonal antibody preparationwhich reacts immunologically with human APE. In other embodiments, thereis provided a monoclonal antibody that reacts immunologically with humanAPE. The monoclonal antibody may farther comprise a detectable label.

In other embodiments, the present invention provides a method fordetermining the premalignant or malignant state of a cell comprisingdetermining the level of APE in the cell, wherein an elevated level ofAPE, as compared to the APE level in a corresponding normal cell,indicates a premalignant or malignant state in the cell.

In particular aspects, the determining comprises the steps of:disrupting the cell; contacting the disrupted cell with an antibody thatreacts immunologically with APE; and quantitating the amount of APEbound to the antibody. In certain embodiments the cell may disrupted bydetergent lysis, freeze-thaw, sonication, osmotic shock or manualrupture. In various independent embodiments the quantitating may be byELISA or by RIA.

In other embodiments, the determining comprises the steps of: isolatingmRNA from the cell; subjecting the mRNA to reverse transcription toproduce cDNA; and quantitating APE cDNA by PCR.

The present invention contemplates a kit for identifying APE levelscomprising: a first antibody that binds immunologically to APE; and anagent for detection of APE bound to the first antibody. In particularembodiments, the agent may comprise a second antibody or polyclonal serathat binds immunologically to an epitope of APE other than that bound bythe first antibody. In other embodiments, the agent may be a secondantibody that binds to the Fc region of the first antibody. In moreparticular aspects the second antibody comprises a detectable label.

The present invention further provides a method for diagnosis ofpremalignant or malignant condition in a human subject which comprises:administering to the subject an imaging agent comprising antibodieswhich react immunologically with APE bound to a label which isdetectable by an external scan of the subject; and externally scanningthe subject to determine whether there is a localized concentration ofthe imaging agent. In certain aspects the label may be a radioactivelabel or may be detectable by an X-ray, positron emission or magneticresonance imaging scanning of the subject.

Also provided by the present invention is a method for therapeutictreatment of an APE-related premalignant or malignant condition in ahuman subject comprising administering to the patient an effectivetherapeutic amount of an agent that reduces the APE activity level inpremalignant or malignant cells of the human subject. In particularembodiments the reducing comprises inhibiting expression of an APE genein the cells. In other embodiments, the reducing comprises inhibitingAPE function in the cells. In certain aspects the inhibiting maycomprise contacting the cells with antisense APE expression constructs.Alternatively, the inhibiting comprises contacting the cells withantibodies that bind immunologically to APE.

It has now been determined that decreased amounts of APE are present inthe cells undergoing and/or likely to undergo apoptosis. This discoveryhas enabled the use of APE as a marker for apoptosis, to which thepresent invention is generally addressed. Thus, in one embodiment, theinvention provides methods and materials for the specific and sensitiveassay of cells to assist in the identification of an apoptotic conditionof the cells, and for modulating the apoptotic behavior of cells. Theinventive methods and materials are expected to be highly useful in manyfields including inter alia in the study and administration of cancerand cancer therapies.

Thus in alternative embodiments, the present invention provides a methodfor identifying apoptosis in a cell comprising (i) obtaining a sampleand (ii) determining the level of APE in the sample, wherein a decreasedlevel of APE, as compared to a normal APE level for a cell of the sametype, indicates that the cell is undergoing apoptosis. In particularembodiments, the level is decreased by at least about 50% compared tothe control. In other embodiments, the level is decreased by at leastabout 75% compared to the control. In yet other embodiments, the levelis decreased by at least about 90% compared to the control.

In preferred embodiments, the cell is a tumor cell that has beensubjected to chemotherapy, radiotherapy or gene therapy. In particularembodiments, the tumor cell is selected from the group consisting ofbrain, lung, liver, spleen, kidney, lymph node, small intestine,pancreas, blood cells, colon, stomach, breast, endometrium, prostate,cervix, testicle, ovary, skin, head and neck, esophagus, bone marrow andblood tumor cells.

In certain aspects of the present invention the determining comprisesevaluating APE protein levels. The determining may comprise evaluatingAPE transcript levels. Alternatively, the evaluating may comprise animmunoassay or quantitative RT-PCR.

The present invention describes a method for monitoring the efficacy ofa cancer therapy comprising (i) administering a therapeutic agent tocancer cells of a subject and (ii) determining the level of APE in acancer cell from the subject, wherein a decreased level of APE, ascompared to the APE level for the cells prior to the administering,indicates that the cell is undergoing apoptosis and the therapy iseffective.

In alternative embodiments, the present invention provides a method fordetermining the apoptotic state of cells in a sample comprising (i)obtaining a sample and (ii) determining the level of APE in cells of thesample, wherein a decreased level of APE, as compared to the APE levelin a cell of the same type, indicates that the cells are undergoingapoptosis. In this context, "a cell of the same type" means a treatedtumor cell as compared to an untreated tumor cell, or a diseased cell ascompared to a normal cell.

In yet another embodiment, there is provided a method for inducingapoptosis in a cell comprising reducing the amount of APE activity inthe cell. In particular embodiments, the reducing comprises inhibitingexpression of an APE gene in the cell. More particularly, the inhibitingcomprises providing to the cell an APE antisense expression construct.In other embodiments, the reducing comprises inhibiting APE function. Incertain embodiments the inhibiting may comprise providing to the cell ananti-APE single-chain antibody expression construct. In otherembodiments, the inhibiting may comprise providing to the cell aninactive APE fragment, peptide or mimetic that competes with APE forbinding to an APE substrate. To the extent that APE has an apoptoticactivity an inactive fragment is defined as an APE fragment that doesnot have such an apoptotic function but retains all other APE-likefunctions.

The present invention, in an alternative embodiment, describes a methodfor inhibiting apoptosis in a cell comprising increasing APE activity inthe cell. In particular, the increasing may comprise providing to thecell APE, or an active fragment thereof. In one embodiment, theproviding comprises contacting the cell with an expression constructencoding APE or an active fragment thereof. In another embodiment, theproviding may comprise contacting the cell with a purified APEpolypeptide. In certain aspects the cell may be a T-cell infected with ahuman immunodeficiency virus.

In another inventive aspect, the present invention provides a method forenhancing the sensitivity of a tumor cell to a chemotherapy, aradiotherapy or gene therapy comprising reducing the amount of APEactivity in the cell. The reducing may comprise inhibiting expression ofan APE gene in the cell. Alternatively, the reducing may compriseinhibiting APE function.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein:

FIG. 1 shows a Western Blot Analysis using affinity purified APEantibody. Total cell extracts (20μg) from the human HPV⁺ cervical linesCaSki, HeLa and SiHa (lanes 2-4 from left, respectively) were run on a12% SDS-polyacrylamide gel, blotted to nitrocellulose and reacted withpurified antibody to the human APE DNA BER repair enzyme. Only a singeprotein band of M_(r) 37,000 was observed. The first lane containedcrude cell extract from E. Coli cells containing the pGET-APE fusionclone. The size of the fuision, overexpressed protein was approximatelyM_(r) 63,000, the predicted size (GST=26,000 and APE=37,000).

FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D, FIG. 2E, FIG. 2F, FIG. 2G and FIG.2H shows APE immunohistochemistry of cervical cancer cell lines. APEantibody staining of HeLa, CaSki, SiHa and C 33a cells is shown in FIG.2A, FIG. 2B, FIG. 2C and FIG. 2D, respectively. The staining of APE isalmost exclusively nuclear. FIG. 2E, FIG. 2F, FIG. 2G and FIG. 2H wereHeLa, CaSki, SiHa and C33a cells reacted with purified IgG from thepreimmune serum at a concentration two times higher than used with theAPE antibody. Little, if any, background staining was detected with thepreimmune staining. Magnification view of all panels was 40×.

FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, FIG. 3E, FIG. 3F, FIG. 3G, FIG. 3Hand FIG. 3I show immunohistochemical staining of normal and CINI (milddysplasia) human cervical tissues. Normal tissue has APE staining innuclei at much lower levels than in the dysplasia tissue. Cells in theCINI tissue have increased levels of APE staining, both in intensity andnumber of cells staining. This increase is mainly in the nuclei,although some cells have increased APE levels in the cytoplasm as well.FIG. 3A and FIG. 3E; hematoxylin and eosin staining of normal cervicaltissue. FIG. 3B; normal tissue with pre-immune antibody. FIG. 3C andFIG. 3D; Normal tissue stained with APE antibody. There are only a fewcells staining in the epithelium of the normal tissue as marked by thearrow. FIG. 3F-FIG. 3I; mild dysplasia (CINI) stained with APE antibody.An increased number of cells are stained between the arrowheads in FIG.3F. Magnification was 10× for FIG. 3B and FIG. 3C, 20× for FIG. 3A, FIG.3D-FIG. 3G and 40× for FIG. 3H and FIG. 3I.

FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, FIG. 4E and FIG. 4F shows animmunohistochemical staining of SCC-G2 and SCC-G3 with human APEantibody. In the SCC tissues, the antibody staining is dramaticallyincreased, both in intensity and numbers of cells staining. FIG. 4A;hematoxylin and eosin staining of CINI tissue with increased number ofnuclei and cell density. FIG. 4B; preimmune control. FIG. 4C, normalcervix tissue with APE antibody. Notice the little or lack of stainingof the gland marked by the arrow. FIG. 4D; SCC-G3 increased number ofcells staining with the APE antibody and increased intensity ofstaining. FIG. 4E; SCC-G3 close up of FIG. 4D (see arrow). Increasedstaining that is both nuclear and cytoplasmic and almost every cell inthis neoplastic region is staining compared to FIG. 4C and the normaltissue in FIG. 3. FIG. 4F; Increased staining of cells in the base ofthe columnar cells (arrow; compare to FIG. 4C). Also, increased cellstaining density in the overall tissue. Magnification was 10× for FIG.4B and FIG. 4D, 20× for FIG. 4A, FIG. 4B and FIG. 4F and 40× for FIG. 4Cand FIG. 4E.

FIG. 5A, FIG. 5B, and FIG. 5C shows immunohistochemical staining ofprostate tissue. FIG. 5A. is hematoxylin and eosin (H&E) staining ofprostate tissue with normal (open arrows) and cancer (closed arrow).FIG. 5B open arrows designate normal glandular tissue with low level ofAPE staining and closed arrow is prostate cancer cells. The level of APEis highly elevated in the prostate cancer. FIG. 5C. cancer cells inprostate invading nerve cell (large arrow).

FIG. 6A and FIG. 6B: Expression of APE in HL-60 cells. FIG. 6A, HL-60cells at 0, 2, 4, and 6 days after treatment with 10₋₅ M RA or 1.25%DMSO, Western blot (top) probed with an affinity purified polyclonalrabbit anti-APE antibody which detects the 37,000 Daltons human APE.Northern blots (middle and bottom) probed for the 1.6 Kb APE mRNA andGAPDH transcripts. FIG. 6B, HL-60 cells treated with 100 nM PMA.

FIG. 7A and FIG. 7B: Expression of APE in HL-60-bcl-2 cells. FIG. 7A,HL-60-bcl-2 cells at 0, 2, 4, and 6 days after treatment with 10₋₅ M RAor 1.25% DMSO, Western blot (top) probed with an affinity purifiedpolyclonal rabbit anti-APE antibody. Northern blots (middle and bottom)probed for the 1.6 Kb APE and GAPDH transcripts. FIG. 7B, HL-60-bcl-2cells treated with 100 nM PMA.

FIG. 8A, FIG. 8B, FIG. 8C and FIG. 8D: Apoptosis in HL-60 (open boxes)and HL-60-bcl-2 (black boxes) cells after differentiation induction.Percent apoptotic cells was determined by counting cells stainingpositive for the TUNNEL reaction. Cells were treated with ethanol(control; FIG. 8A), RA (FIG. 8B), DMSO (FIG. 8C), or PMA (FIG. 8D).Error bars indicate the mean standard deviation, and * indicates p value<0.05.

FIG. 9A, FIG. 9B and FIG. 9C: Specificity of APE expressiondown-regulation and apoptosis. FIG. 9A 50:50 mix of untreated HL-60cells and HL-60 cells treated with RA for 6 days. FIG. 9B same cells asin FIG. 9A stained with rhodamine-labeled (re) antihuman APE. FIG. 9Csame cells as FIG. 9A stained for fragmented DNA using fluoresceinlabeled (green) and TUNNEL assay. Individual cell identified with arrowsdisplay relative APE expression and fragmented DNA in the same cells forcomparison.

FIG. 10A and FIG. 10B: Enhanced viability of HL-60-bcl-2 granulocytes.HL-60 and HL-60-bcl-2 cells were induced with RA (1 μmol/L) which wasadded at day 0. At the indicated days after RA treatment (FIG. 10A),cell number was determined using a hemocytometer chamber and (FIG. 10B)percentage of cell viability was determined by trypan blue dyeexclusion. The indicated points represent the mean of triplicateexperiments (▴) HL-60 wt; (∘) HL-60 Bcl2.

FIG. 11A and FIG. 11B: Reduced DNA fragmentation in induced HL-60-bcl-2granulocytes. HL-60 and HL-60-bcl-2 cells were induced with RA (1μmol/L) which was added at day 0. FIG. 11A At the indicated days afterRA treatment, the TUNNEL assay was used to quantitate the percentage ofRA-induced cells harboring detectable DNA fragments. FIG. 11B Atindicated days RA treatment, DNA fragmentation was quantitated bydiphenylamine assay. The ratio of DNA cleavage products to highmolecular weight DNA is given as percentage (▴) HL-60 wt; (∘) HL-60Bcl2.

FIG. 12A and FIG. 12B. Northern and Western blot analyses of APEexpression in CD34 cells induced to differentiate down the lymphoid andmyeloid pathways

FIG. 13: Schematic representation of retroviral constructs containingthe human APE cDNA.

FIG. 14: Additional APE constructs.

FIG. 15: Schematic of base excision repair (BER) pathway indicating therole of APE in the BER pathway.

FIG. 16: Polyclonal antibody produced in rabbit or chicken to the humanAPE protein. Western blot analysis of HeLa cell (human) or NIH3T3(mouse) cells using antibody produced in rabbits or chickens. Theantibodies were affinity purified and only a single cross-reactingprotein is visible in the human or mouse cells.

FIG. 17: Illustration of the DNA repair domain of APE. The DNA repairregion is located in the carboxy terminal of the protein. Comparison ofAPE regions of hAPE with APE from A. thaliana (Arp), D. melanogaster(Rip1), S. pneumoniae (ExoA), E. coli (exoIII).

FIG. 18A, FIG. 18B and FIG. 18C. Prostate tissue with APE. FIG. 18Alower magnification of a section of prostate tissue showing normal(arrowheads) and cancer cells (arrows with tails). FIG. 18B highermagnification of cancer cells from FIG. 18A. Strong nuclear staining ina punctuate manner. FIG. 18C. low power magnification of prostate tissuewith cancerous region to lower left (below arrows) and normal cells inupper right region. Distinctive higher levels of APE staining in thecancerous region.

FIG. 19A, and FIG. 19B. Nuclear and cytoplasmic staining of prostatecancer cells. FIG. 19A H&E staining of prostate with cancer cells. FIG.19B prostate cancer cells demonstrating both nuclear (arrowhead) andcytoplasmic (arrows with tail) staining with APE antibody, both athigher levels than in normal tissue.

FIG. 20. A comparison of the efficacy of the present APE polyclonal to acommercially available polyclonal and five monoclonal antibodies thathave not yet been published.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

For the purpose of promoting an understanding of the principles of theinvention, reference will now be made to certain preferred embodimentsthereof and specific language will be used to describe the same. It willnevertheless be understood that no limitation of the scope of theinvention is thereby intended, such alterations, further modificationsand applications of the principles of the invention as described hereinbeing contemplated as would normally occur to one skilled in the art towhich the invention relates.

I. THE PRESENT INVENTION

The present invention involves the association of APE with(pre)malignant states. Furthermore, the present inventor has discoveredthat APE can be used as a specific marker for apoptotic conditions incells. Thus, APE level is an important indicator of the growth status ofa cell. These discoveries will be exploited in the diagnosis, treatmentand monitoring of malignancies and apoptotic conditions, and arediscussed in further detail herein below.

A. APE is Increased in (Pre)malignant States

In the course of studies characterizing a squamous cell carcinoma of theuterine cervix, it was discovered that greater amounts of APE werepresent in these tumor cells. This observation permits the use of APE asa marker for (pre)malignant conditions in human subjects, to which thepresent invention is generally addressed. More specifically, it has beendiscovered that apurinic/apyriridinic endonucleases (APE) can be used asspecific markers for premalignant and malignant conditions in humans.Thus, in one embodiment, the present invention provides both methods anddevices which facilitate the diagnosis of a (pre)malignant condition ina human subject, which employ APE as a marker for the condition.

In another aspect of the invention, there is contemplated the treatmentof APE-related malignancies where abnormally high APE levels not onlyare indicative of cancer, but are responsible, to some extent, for themalignant phenotype. Thus, by targeting the overexpression of APE and/orAPE function in cells with a therapeutic regimen, it will be possible toblock or inhibit the abnormally high activity of APE and thereby restorenormal growth patterns to the cell. This approach may prove particularlyadvantageous in combination with other chemo-, radio- or gene-basedtherapies.

B. APE is Inversely Related to the Apoptotic State

It further has been discovered by the present inventor thatapurinic/apyrimidinic endonucleases (APE) can be used as a specificmarker for apoptotic conditions in cells. More specifically, the presentinvention shows that decreased levels of APE are indicative of cellsthat are undergoing apoptosis. For example, in work to date, the overalllevels of APE in blood cells, in particular, cells of lymphoid andmyeloid lineage, have been substantially reduced relative to controls asthese cells differentiate and apoptose. The present invention may beapplied readily to other cell types, including any cells that naturallyundergo apoptosis, for example, cells involved in normal or abnormaldevelopmental aging where apoptosis occurs. The present inventor hasdetermined that APE levels decline well before a positive signal isobserved using the TUNNEL assay, an assay commonly used to determineapoptosis (Hockenbery, 1995). Thus, methods involving the use of APE toidentify cells destined to die via apoptosis constitute an improvementover those currently available.

Thus, in another aspect, the present invention provides methods whichfacilitate the determination of whether a cell is undergoing apoptosis,using diminution of APE levels as an indicator of this condition. Manycancer chemotherapeutic agents act by inducing cancer cells to undergoapoptosis. Thus, in a particular embodiment, declining levels of APEwill be used to identify cells undergoing apoptosis as part of atherapeutic regiment. This will permit one to monitor the efficacy of atreatment and, in certain cases, stop treatment where the effects are orare not seen, thereby avoiding treatment related toxicity.

In yet another aspect, the inventor discloses the possible involvementof APE in the regulation of various topoisomerase II function. Manyanticancer drugs "poison" topoisomerase II by enhancing itsdouble-stranded DNA cleavage activity. AP sites are position-specifictopoisomerase II poisons (Kingma and Osheroff, 1997). As such,downregulation of APE according to the present invention, in combinationconventional topoisomerase II-targeted anticancer drugs, may proveparticularly useful against cancers involving topoisomerase IIaberrations, and may help overcome multidrug resistance in such cancers.

In still other aspects of the invention, it is expected that thesusceptibility of cells to apoptosis may be modulated by increasing, ordecreasing the amount of APE enzymatic activity or protein in the cells,to achieve a respective decrease or increase in apoptosis orsusceptibility to apoptosis. Thus, further embodiments of the inventionrelate to such methods for modulating cellular susceptibility toapoptosis. This may be exploited by causing cells to be sensitized tocertain classic radio- or chemotherapeutic agents. The decrease orincrease in APE activity or protein can be achieved in any suitablefashion, including for example, by action upon the protein per se (e.g.,to deactivate the protein), by antibodies or other means, or by actionupon transcription or translation of APE, e.g., by antisenseoligonucleotides. In this regard, as indicated above, the major APE genehas been cloned and its sequence and location are known. These and otheraspects of the present invention are presented in further detail hereinbelow.

In yet another aspect, it may be observed that some cells undergo apremature cell death. These cells, whether responding to an externalstress or reacting to an internal genetic abnormality, may nonethelessremain finctional up to their demise. In such cases, it may beadvantageous to promote APE such that "normal" APE expression isachieved. This may serve to delay or even prevent apoptosis in thesecells.

II. PROTEINS

According to the present invention, APE has been identified as aneffective marker for (pre)malignant conditions. The inventor has foundthat the levels of APE in dysplasia, carcinoma in situ and squamous cellcarcinomas of the cervix and the prostate have been substantiallyelevated relative to controls. This discovery will be exploited in avariety of sampling, prognostic and monitorial strategies in the contextof the present invention. In particular embodiments, the presentinvention isolates APE from a variety of sources in order to makeantibodies for use in these strategies.

According to the present invention, APE has been identified as a markerof induction of apoptosis. The inventor has observed an increase inapoptosis in cells having decreased levels of APE. The uses stemmingfrom this observation are manifold, as described above, and will beexploited in the context of the present invention. In one aspect of theinvention, therefore, use of the APE protein is contemplated. Forexample, one may seek to generate antibodies reactive with this moleculefor use in certain assays. In other embodiments, one may wish to makefunctional or non-functional variants of this molecule to augment orinhibit APE function in vivo.

Thus, in addition to use of the entire, wild-type APE molecule, thepresent invention also relates to variants and fragments of thepolypeptide that may or may not retain the normal functions of APE.Fragments including the N-terminus of the molecule may be generated bygenetic engineering of translation stop sites within the coding region(discussed below). Alternatively, treatment of the APE molecule withproteolytic enzymes, known as protease, can produces a variety ofN-terminal, C-terminal and internal fragments. Examples of fragments mayinclude contiguous residues of the APE sequence given in SEQ ID NO:2, of6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 30, 35, 40, 45, 50, 55, 60, 65, 75, 80, 85, 90, 95, 100, 200, 300,400 or more amino acids in length. These fragments may be purifiedaccording to known methods, such as precipitation (e.g., ammoniumsulfate), HPLC, ion exchange chromatography, affinity chromatography(including immunoaffinity chromatography) or various size separations(sedimentation, gel electrophoresis, gel filtration).

A. The APE Polypeptide Functional Aspects

The major human APE is a known protein of about 37,000 Daltons. Thecorresponding gene has been cloned and expressed (cDNA sequencedeposited in the GenBank data base, Accession No. M80261, as reported inDemple et al., (1991)). APE acts on AP sites in DNA and stimulates theDNA binding activity of Jun-Jun and Fos-Jun dimers, as well as a numberof other transcription factors such as NFKB, Myb, AP-1 proteins andmembers of the ATF/CREB family.

The DNA binding activity of these molecules is sensitive toreduction-oxidation (redox). APE, which is responsible for the majorAP-1 redox activity in HeLa cells, represents a novel redox component ofthe signal transduction processes that regulate eukaryotic geneexpression. APE also has been shown to regulate the activity of p53through a redox mechanism. Since p53 is thought to mediate and controlinteraction between transcription and DNA repair signaling followinggenomic damage, APE may also be an important member of this regulatorypathway. Furthermore, redox regulation via APE expression and activitymay limit the total amount of functional Fos-Jun complexes and regulatethe transformation (cancer promoting) activity of these proteins.Therefore, APE may form a unique link between the DNA base excisionrepair pathway (FIG. 15), oxidative signaling, transcription factorregulation and cell-cycle control.

The present invention has correlated APE level changes with cancerphenotypes. It is also of interest that APE is also involved inregulating the redox state of various proteins, including transcriptionfactors such as p53 as discussed above. In some human cancers mutationsaffect the ability of proteins to have their redox status modified byproteins like APE. Hence it may be useful to look at the redox state ofAPE targets. It may also be advantageous to look at mutations in theredox domain of APE which affect its ability to modulate other proteins.If APE redox activity is destroyed, it doesn't matter if the otherdownstream proteins, such as p53 or other redox regulated proteins, aremutated or not as they may not be able to be reduced or oxidized due tothe APE activity.

B. APE Polypeptide Structural Aspects

The gene for APE encodes a 318 amino acid polypeptide (SEQ ID NO:2). Thepredicted molecular weight of this molecule is 37,000 Daltons. The DNArepair domain is located in the carboxy 80% of the protein with aminoacids Asp(283), Asp (308) and His (309) being involved in the repairactive site. Glu (96) is also important for repair activity (FIG. 17).The redox domain is in the amino portion of the APE protein with Cys(65)being crucial for redox activity (Barzilay, 1995a; Barzilay, 1995b)

As discussed below, the APE gene can be inserted into an appropriateexpression system. The gene can be expressed in any number of differentrecombinant DNA expression systems to generate large amounts of thepolypeptide product, which can then be purified and used to vaccinateanimals to generate antisera with which further studies may beconducted.

Amino acid sequence variants of the polypeptide may be prepared. Thesemay, for instance, be minor sequence variants of the polypeptide thatarise due to natural variation within the population or they may behomologues found in other species. The variations may or may not affectthe function of the molecule, and similarly, they may or may not affectthe immunological properties of the molecule. Sequence variants can beprepared by standard methods of site-directed mutagenesis such as thosedescribed below in the following section.

C. Variants of APE

Amino acid sequence variants of the polypeptide can be substitutional,insertional or deletion variants. Deletion variants lack one or moreresidues of the native protein which are not essential for function orinununogenic activity, and are exemplified by the variants lacking atransmembrane sequence described above. Another common type of deletionvariant is one lacking secretory signal sequences or signal sequencesdirecting a protein to bind to a particular part of a cell. Insertionalmutants typically involve the addition of material at a non-terminalpoint in the polypeptide. This may include the insertion of animmunoreactive epitope or simply a single residue. Terminal additions,called fusion proteins, are discussed below.

Substitutional variants typically contain the exchange of one amino acidfor another at one or more sites within the protein, and may be designedto modulate one or more properties of the polypeptide, such as stabilityagainst proteolytic cleavage, without the loss of other functions orproperties. Substitutions of this kind preferably are conservative, thatis, one amino acid is replaced with one of similar shape and charge.Conservative substitutions are well known in the art and include, forexample, the changes of: alanine to serine; arginine to lysine;asparagine to glutamine or histidine; aspartate to glutamate; cysteineto serine; glutamine to asparagine; glutamate to aspartate; glycine toproline; histidine to asparagine or glutamine; isoleucine to leucine orvaline; leucine to valine or isoleucine; lysine to arginine; methionineto leucine or isoleucine; phenylalanine to tyrosine, leucine ormethionine; serine to threonine; threonine to serine; tryptophan totyrosine; tyrosine to tryptophan or phenylalanine; and valine toisoleucine or leucine.

The following is a discussion based upon changing of the amino acids ofa protein to create an equivalent, or even an improved,second-generation molecule. For example, certain amino acids may besubstituted for other amino acids in a protein structure withoutappreciable loss of interactive binding capacity with structures suchas, for example, antigen-binding regions of antibodies or binding siteson substrate molecules. Since it is the interactive capacity and natureof a protein that defines that protein's biological functional activity,certain amino acid substitutions can be made in a protein sequence, andits underlying DNA coding sequence, and nevertheless obtain a proteinwith like properties. It is thus contemplated by the inventor thatvarious changes may be made in the DNA sequences of genes withoutappreciable loss of their biological utility or activity, as discussedbelow. Table 1 shows the codons that encode particular amino acids.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art kyte & Doolittle, 1982). It is accepted that therelative hydropathic character of the amino acid contributes to thesecondary structure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis oftheir hydrophobicity and charge characteristics (Kyte & Doolittle,1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8);tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2);glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5);lysine (-3.9); and arginine (-4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e., still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein. As detailed in U.S. Pat. No. 4,554,101, thefollowing hydrophilicity values have been assigned to amino acidresidues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate(+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine(0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine*-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine(-1.8); isoleucine (1.8); tyrosine (-2.3); phenylalanine (-2.5);tryptophan (-3.4).

It is understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity value and still obtain a biologicallyequivalent and immunologically equivalent protein. In such changes, thesubstitution of amino acids whose hydrophilicity values are within ±2 ispreferred, those that are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally based on therelative similarity of the amino acid side-chain substituents, forexample, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions that take various of the foregoingcharacteristics into consideration are well known to those of skill inthe art and include: arginine and lysine; glutamate and aspartate;serine and threonine; glutamine and asparagine; and valine, leucine andisoleucine.

Another embodiment for the preparation of polypeptides according to theinvention is the use of peptide mimetics. Mimetics arepeptide-containing molecules that mimic elements of protein secondarystructure. See, for example, Johnson et al., "Peptide Turn Mimetics" inBIOTECHNOLOGY AND PHARMACY, Pezzuto et al., Eds., Chapman and Hall, NewYork (1993). The underlying rationale behind the use of peptide mimeticsis that the peptide backbone of proteins exists chiefly to orient aminoacid side chains in such a way as to facilitate molecular interactions,such as those of antibody and antigen. A peptide mimetic is expected topermit molecular interactions similar to the natural molecule. Theseprinciples may be used, in conjunction with the principles outlineabove, to engineer second generation molecules having many of thenatural properties of APE, but with altered and even improvedcharacteristics.

D. Fusion Proteins

A specialized kind of insertional variant is the fusion protein. Thismolecule generally has all or a substantial portion of the nativemolecule, linked at the N- or C-terminus, to all or a portion of asecond polypeptide. For example, fusions typically employ leadersequences from other species to permit the recombinant expression of aprotein in a heterologous host. Another useful fusion includes theaddition of a immunologically active domain, such as an antibodyepitope, to facilitate purification of the fusion protein. Inclusion ofa cleavage site at or near the fusion junction will facilitate removalof the extraneous polypeptide after purification. Other useful fusionsinclude linking of functional domains, such as active sites fromenzymes, glycosylation domains, cellular targeting signals ortransmembrane regions.

E. Purification of Proteins

It will be desirable to purify APE, APE fragments or variants thereof.Protein purification techniques are well known to those of skill in theart. These techniques involve, at one level, the crude fractionation ofthe cellular milieu to polypeptide and non-polypeptide fractions. Havingseparated the polypeptide from other proteins, the polypeptide ofinterest may be further purified using chromatographic andelectrophoretic techniques to achieve partial or complete purification(or purification to homogeneity). Analytical methods particularly suitedto the preparation of a pure peptide are ion-exchange chromatography,exclusion chromatography; polyacrylamide gel electrophoresis;isoelectric focusing. A particularly efficient method of purifyingpeptides is fast protein liquid chromatography or even HPLC.

Certain aspects of the present invention concern the purification, andin particular embodiments, the substantial purification, of an encodedprotein or peptide. The term "purified protein or peptide" as usedherein, is intended to refer to a composition, isolatable from othercomponents, wherein the protein or peptide is purified to any degreerelative to its naturally-obtainable state. A purified protein orpeptide therefore also refers to a protein or peptide, free from theenvironment in which it may naturally occur.

Generally, "purified" will refer to a protein or peptide compositionthat has been subjected to fractionation to remove various othercomponents, and which composition substantially retains its expressedbiological activity. Where the term "substantially purified" is used,this designation will refer to a composition in which the protein orpeptide forms the major component of the composition, such asconstituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95% or more of the proteins in the composition.

Various methods for quantifying the degree of purification of theprotein or peptide will be known to those of skill in the art in lightof the present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis. A preferred methodfor assessing the purity of a fraction is to calculate the specificactivity of the fraction, to compare it to the specific activity of theinitial extract, and to thus calculate the degree of purity, hereinassessed by a " purification factor!-fold" (e.g. 2-fold, 4-fold, 8-fold,10-fold, 25-fold, 100-fold or more). The actual units used to representthe amount of activity will, of course, be dependent upon the particularassay technique chosen to follow the purification and whether or not theexpressed protein or peptide exhibits a detectable activity.

Various techniques suitable for use in protein purification will be wellknown to those of skill in the art. These include, for example,precipitation with ammonium sulphate, PEG, antibodies and the like or byheat denaturation, followed by centrifugation; chromatography steps suchas ion exchange, gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of such and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

There is no general requirement that the protein or peptide always beprovided in their most purified state. Indeed, it is contemplated thatless substantially purified products will have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme. For example, it is appreciatedthat a cation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater "-fold" purification thanthe same technique utilizing a low pressure chromatography system.Methods exhibiting a lower degree of relative purification may haveadvantages in total recovery of protein product, or in maintaining theactivity of an expressed protein.

It is known that the migration of a polypeptide can vary, sometimessignificantly, with different conditions of SDS/PAGE (Capaldi et al.,1977). It will therefore be appreciated that under differingelectrophoresis conditions, the apparent molecular weights of purifiedor partially purified expression products may vary.

High Performance Liquid Chromatography (HPLC) is characterized by a veryrapid separation with extraordinary resolution of peaks. This isachieved by the use of very fine particles and high pressure to maintainan adequate flow rate. Separation can be accomplished in a matter ofminutes, or at most an hour. Moreover, only a very small volume of thesample is needed because the particles are so small and close-packedthat the void volume is a very small fraction of the bed volume. Also,the concentration of the sample need not be very great because the bandsare so narrow that there is very little dilution of the sample.

Gel chromatography, or molecular sieve chromatography, is a special typeof partition chromatography that is based on molecular size. The theorybehind gel chromatography is that the column, which is prepared withtiny particles of an inert substance that contain small pores, separateslarger molecules from smaller molecules as they pass through or aroundthe pores, depending on their size. As long as the material of which theparticles are made does not adsorb the molecules, the sole factordetermining rate of flow is the size. Hence, molecules are eluted fromthe column in decreasing size, so long as the shape is relativelyconstant. Gel chromatography is unsurpassed for separating molecules ofdifferent size because separation is independent of all other factorssuch as pH, ionic strength, temperature, etc. There also is virtually noadsorption, less zone spreading and the elution volume is related in asimple matter to molecular weight.

Affinity Chromatography is a chromatographic procedure that relies onthe specific affinity between a substance to be isolated and a moleculethat it can specifically bind to. This is a receptor-ligand typeinteraction. The column or membrane-bound material is synthesized bycovalently coupling one of the binding partners to an insoluble matrixor membrane. The column material is then able to specifically adsorb thesubstance from the solution. Elution occurs by changing the conditionsto those in which binding will not occur (alter pH, ionic strength,temperature, etc.).

The matrix should be a substance that itself does not adsorb moleculesto any significant extent and that has a broad range of chemical,physical and thermal stability. The ligand should be coupled in such away as to not affect its binding properties. The ligand should alsoprovide relatively tight binding. And it should be possible to elute thesubstance without destroying the sample or the ligand. One of the mostcommon forms of affinity chromatography is immunoaffinitychromatography. The generation of antibodies that would be suitable foruse in accord with the present invention is discussed below.

F. Synthetic Peptides

The present invention also describes smaller APE-related peptides foruse in various embodiments of the present invention. Because of theirrelatively small size, the peptides of the invention can also besynthesized in solution or on a solid support in accordance withconventional techniques. Various automatic synthesizers are commerciallyavailable and can be used in accordance with known protocols. See, forexample, Stewart and Young, (1984); Tam et al., (1983); Merrifield,(1986); and Barany and Merrifield (1979), each incorporated herein byreference. Short peptide sequences, or libraries of overlappingpeptides, usually from about 6 up to about 35 to 50 amino acids, whichcorrespond to the selected regions described herein, can be readilysynthesized and then screened in screening assays designed to identifyreactive peptides. Alternatively, recombinant DNA technology may beemployed wherein a nucleotide sequence which encodes a peptide of theinvention is inserted into an expression vector, transformed ortransfected into an appropriate host cell and cultivated underconditions suitable for expression.

G. Antigen Compositions

As stated above, the present invention provides, in one embodiment, forthe use of APE proteins or peptides as antigens for the immunization ofanimals relating to the production of antibodies. APE, or portionsthereof, may be coupled, bonded, bound, conjugated or chemically-linkedto one or more agents via linkers, polylinkers or derivatized aminoacids. This may be performed such that a bispecific or multivalentcomposition or vaccine is produced. In a particular embodiment, theantigen composition comprises an APE-fusion protein. The production ofantibodies using this composition is described in Example 1 hereinbelow. It is further envisioned that other methods may be used in thepreparation of APE antibodies, as will be familiar to those of skill inthe art.

III. NUCLEIC ACIDS

As disclosed above, the present invention provides, in one aspect, a DNAsequence encoding an APE protein. In certain aspects, this DNA may beuseful in diagnosis of premalignant conditions. In other aspects, thisDNA will be useful in the diagnosis of apoptotic conditions. Indeed,nucleic acids are contemplated to be useful for the expression of APEprotein, fragments or variants for a variety of diagnostic ortherapeutic purposes related independently to (pre)malignant andapoptotic states of a cell. Thus, the present invention also encompassesexpression vectors designed to provide for the production of APE. Inother aspects, it may be advantageous to decrease the production of APE.This may be accomplished, in one embodiment, by employing antisense APEconstructs.

In this regard, as used herein, "DNA sequence" refers to a DNA polymer,in the form of a separate fragment or as a component of a larger DNAconstruct. Such sequences are preferably provided in the form of an openreading frame uninterrupted by internal nontranslated sequences, orintrons, which are typically present in eukaryotic genes. Genomic DNAcontaining the relevant sequences could also be used. Sequences ofnon-translated DNA may be present 5' or 3' from the open reading frame,where the same do not interfere with manipulation or expression of thecoding regions.

The gene for the primary human APE has been identified and is known tothose of skill in the art. The present invention is not limited in scopeto this gene, however, as one of ordinary skill in the could, using thepresent disclosure, identify and employ homologs from various otherspecies (e.g., rat, rabbit, monkey, gibbon, chimp, ape, baboon, cow,pig, horse, sheep, cat and other species) to achieve the goals outlinedherein.

In addition, it should be clear that the present invention is notlimited to the specific nucleic acids disclosed herein. As discussedbelow, an "APE gene" may contain a variety of different bases and yetstill produce polypeptides that are structurally and/or functionallyindistinguishable, from the human gene disclosed herein.

Similarly, any reference to a nucleic acid should be read asencompassing an expression vector and host cell containing that nucleicacid. In addition to diagnostic considerations, cells expressing nucleicacids of the present invention may prove particularly useful in thecontext of screening for agents that induce, repress, inhibit, augment,interfere with, block, abrogate, stimulate or enhance the function ofthe APE polypeptide.

A. Nucleic Acids Encoding APE

The human gene for APE is disclosed in SEQ ID NO:1. Nucleic acidsaccording to the present invention may encode an entire APE gene, afunctional domain of an APE gene, or any other fragment of the APEsequence set forth herein. The nucleic acid may be derived from genomicDNA, i.e., cloned directly from the genome of a particular organism. Inpreferred embodiments, however, the nucleic acid would comprisecomplementary DNA (cDNA). Also contemplated is a cDNA plus a naturalintron or an intron derived from another gene; such engineered moleculesare sometime referred to as "mini-genes." At a minimum, these and othernucleic acids of the present invention may be used as molecular weightstandards in, for example, gel electrophoresis.

The term "cDNA" is intended to refer to DNA prepared using messenger RNA(mRNA) as template. The advantage of using a cDNA, as opposed to genomicDNA or DNA polymerized from a genomic, non- or partially-processed RNAtemplate, is that the cDNA primarily contains coding sequences of thecorresponding protein. There may be times when the full or partialgenomic sequence is preferred, such as where the non-coding regions arerequired for optimal expression or where non-coding regions such asintrons are to be targeted in an antisense strategy.

As stated above, it also is contemplated that a given APE from a givenspecies may be represented by natural variants that have slightlydifferent nucleic acid sequences but, nonetheless, encode the sameprotein (see Table 1 below).

As used in this application, the term "a nucleic acid encoding an APEpolypeptide" refers to a nucleic acid molecule that has been isolatedfree of total cellular nucleic acid. In preferred embodiments, theinvention concerns a nucleic acid sequence essentially as set forth inSEQ ID NO:1. The term "as set forth in SEQ ID NO:1" means that thenucleic acid sequence substantially corresponds to a portion of SEQ IDNO:1. The term "functionally equivalent codon" is used herein to referto codons that encode the same amino acid, such as the six codons forarginine or serine (Table 1, below), and also refers to codons thatencode biologically equivalent amino acids, as discussed in thefollowing pages.

                  TABLE 1    ______________________________________    Amino Acids    Codons    ______________________________________    Alanine   Ala    A     GCA  GCC  GCG  GCU    Cysteine  Cys    C     UGC  UGU    Aspartic acid              Asp    D     GAC  GAU    Glutamic acid              Glu    E     GAO  GAG    Phenylalanine              Phe    F     UUC  UUU    Glycine   Gly    G     GGA  GGC  GGG  GGU    Histidine His    H     CAC  CAU    Isoleucine              Ile    I     AUA  AUC  AUU    Lysine    Lys    K     AAA  AAG    Leucine   Leu    L     UUA  UUG  CUA  CUC  CUG  CUU    Methionine              Met    M     AUG    Asparagine              Asn    N     AAC  AAU    Proline   Pro    P     CCA  CCC  CCG  CCU    Glutamine Gln    Q     CAA  CAG    Arginine  Arg    R     AGA  AGG  CGA  CGC  CGG  CGU    Serine    Ser    S     AGC  AGU  UCA  UCC  UCG  UCU    Theronine Thr    T     ACA  ACC  ACG  ACU    Valine    Val    V     GUA  GUC  GUG  GUU    Tryptophan              Trp    W     UGG    Tyrosine  Tyr    Y     UAC  UAU    ______________________________________

Allowing for the degeneracy of the genetic code, sequences that have atleast about 50%, usually at least about 60%, more usually about 70%,most usually about 80%, preferably at least about 90% and mostpreferably about 95% of nucleotides that are identical to thenucleotides of SEQ ID NO:1 will be sequences that are "as set forth inSEQ ID NO:1". Sequences that are essentially the same as those set forthin SEQ ID NO:1 may also be functionally defined as sequences that arecapable of hybridizing to a nucleic acid segment containing thecomplement of SEQ ID NO:1 under standard conditions.

The DNA segments of the present invention include those encodingbiologically functional equivalent APE proteins and peptides, asdescribed above. Such sequences may arise as a consequence of codonredundancy and amino acid functional equivalency that are known to occurnaturally within nucleic acid sequences and the proteins thus encoded.Alternatively, functionally equivalent proteins or peptides may becreated via the application of recombinant DNA technology, in whichchanges in the protein structure may be engineered, based onconsiderations of the properties of the amino acids being exchanged.Changes designed by man may be introduced through the application ofsite-directed mutagenesis techniques or may be introduced randomly andscreened later for the desired function, as described below.

DNA analog sequences are "substantially identical" to the specific DNAsequences disclosed herein if: (a) the DNA analog sequence is derivedfrom substantially the entire coding regions of the native mammalian APEgene; or (b) the DNA analog sequence is comparable in length with andcapable of hybridization to DNA sequences of (a) under moderatelystringent conditions and which encode biologically active APE molecules;or (c) DNA sequences which are degenerated as a result of the geneticcode to the DNA analog sequences defined in (a) or (b) and which encodebiologically active APE molecules. Substantially identical analogproteins will be greater than about 80 percent similar to thecorresponding sequence of the native protein. Sequences having lesserdegrees of similarity but comparable biological activity are consideredto be equivalents. In defining nucleic acid sequences, all subjectnucleic acid sequences capable of encoding substantially similar aminoacid sequences are considered substantially similar to a referencenucleic acid sequence.

B. Oligonucleotide Probes and Primers

Naturally, the present invention also encompasses DNA segments that arecomplementary, or essentially complementary, to the sequence set forthin SEQ ID NO: 1. Nucleic acid sequences that are "complementary" arethose that are capable of base-pairing according to the standardWatson-Crick complementary rules. As used herein, the term"complementary sequences" means nucleic acid sequences that aresubstantially complementary, as may be assessed by the same nucleotidecomparison set forth above, or as defined as being capable ofhybridizing to the nucleic acid segment of SEQ ID NO:1 under relativelystringent conditions such as those described herein. Such sequences mayspan the entire coding region or fragments thereof.

Alternatively, the hybridizing segments may be relatively shortoligonucleotides. Sequences of 17 bases long should occur only once inthe human genome and, therefore, suffice to specify a unique targetsequence. Although shorter oligomers are easier to make and increase invivo accessibility, numerous other factors are involved in determiningthe specificity of hybridization. Both binding affinity and sequencespecificity of an oligonucleotide to its complementary target increaseswith increasing length. It is contemplated that exemplaryoligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or morebase pairs will be used, although others are contemplated.

Longer polynucleotides having 250, 500, 1000 bases or longer arecontemplated as well. Such polynucleotides will find use, for example,as probes in Southern and Northern blots and as primers in amplificationreactions.

Suitable hybridization conditions will be well known to those of skillin the art. In certain applications, for example, substitution of aminoacids by site-directed mutagenesis, it is appreciated that lowerstringency conditions are required. Under these conditions,hybridization may occur even though the sequences of probe and targetstrand are not perfectly complementary, but are mismatched at one ormore positions. Conditions may be rendered less stringent by increasingsalt concentration and decreasing temperature. For example, a mediumstringency condition could be provided by about 0.1 to 0.25M NaCl attemperatures of about 37° C. to about 55° C., while a low stringencycondition could be provided by about 0.15M to about 0.9M salt, attemperatures ranging from about 20° C. to about 55° C. Thus,hybridization conditions can be readily manipulated, and thus willgenerally be a method of choice depending on the desired results.

In other embodiments, hybridization may be achieved under conditions of,for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl,3 mM MgCl₂, 10 mMdithiothreitol, at temperatures between approximately 20° C. to about37° C. Other hybridization conditions utilized could includeapproximately 10 mM Tris-HCl (pH 8.3), 50 mM KC,1, 1.5 μM MgCl₂, attemperatures ranging from approximately 40° C. to about 72° C. Formamideand SDS also may be used to alter the hybridization conditions.

One way of exploiting probes and primers of the present invention is insitedirected, or site-specific mutagenesis. Site-specific mutagenesis isa technique useful in the preparation of individual peptides, orbiologically functional equivalent proteins or peptides, throughspecific mutagenesis of the underlying DNA. The technique furtherprovides a ready ability to prepare and test sequence variants,incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into the DNA.Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered.

The technique typically employs a bacteriophage vector that exists inboth a single stranded and double stranded form. Typical vectors usefulin site-directed mutagenesis include vectors such as the M13 phage.These phage vectors are commercially available and their use isgenerally well known to those skilled in the art. Double strandedplasmids are also routinely employed in site directed mutagenesis, whicheliminates the step of transferring the gene of interest from a phage toa plasmid.

In general, site-directed mutagenesis is performed by first obtaining asingle-stranded vector, or melting of two strands of a double strandedvector which includes within its sequence a DNA sequence encoding thedesired protein. An oligonucleotide primer bearing the desired mutatedsequence is synthetically prepared. This primer is then annealed withthe single-stranded DNA preparation, taking into account the degree ofmismatch when selecting hybridization conditions, and subjected to DNApolymerizing enzymes such as E. coli polymerase I Klenow fragment, inorder to complete the synthesis of the mutation-bearing strand. Thus, aheteroduplex is formed wherein one strand encodes the originalnon-mutated sequence and the second strand bears the desired mutation.This heteroduplex vector is then used to transform appropriate cells,such as E. coli cells, and clones are selected that include recombinantvectors bearing the mutated sequence arrangement.

The preparation of sequence variants of the selected gene usingsite-directed mutagenesis is provided as a means of producingpotentially useful species and is not meant to be limiting, as there areother ways in which sequence variants of genes may be obtained. Forexample, recombinant vectors encoding the desired gene may be treatedwith mutagenic agents, such as hydroxylamine, to obtain sequencevariants.

C. Antisense Constructs

In some cases, it will be desirable to decrease levels of APE, in thetherapeutic treatment of cancer cells. In other cases decreases in APEexpression will be important, for example, to facilitate an increase inapoptosis or to enhance the efficacy of a conventional therapy.Antisense treatments are one way of accomplishing such a decrease in APElevels and expression. Antisense technology also may be used to"knock-out" function of APE in the development of cell lines ortransgenic mice for research, diagnostic and screening purposes.

Antisense methodology takes advantage of the fact that nucleic acidstend to pair with "complementary" sequences. By complementary, it ismeant that polynucleotides are those which are capable of base-pairingaccording to the standard Watson-Crick complementary rules. That is, thelarger purines will base pair with the smaller pyrimidines to formcombinations of guanine paired with cytosine (G:C) and adenine pairedwith either thymine (A:T) in the case of DNA, or adenine paired withuracil (A:U) in the case of RNA. Inclusion of less common bases such asinosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others inhybridizing sequences does not interfere with pairing.

Targeting double-stranded (ds) DNA with polynucleotides leads totriple-helix formation; targeting RNA will lead to double-helixformation. Antisense polynucleotides, when introduced into a targetcell, specifically bind to their target polynucleotide and interferewith transcription, RNA processing, transport, translation and/orstability. Antisense RNA constructs, or DNA encoding such antisenseRNA's, may be employed to inhibit gene transcription or translation orboth within a host cell, either in vitro or in vivo, such as within ahost animal, including a human subject.

Antisense constructs may be designed to bind to the promoter and othercontrol regions, exons, introns or even exon-intron boundaries of agene. It is contemplated that the most effective antisense constructswill include regions complementary to intron/exon splice junctions.Thus, it is proposed that a preferred embodiment includes an antisenseconstruct with complementarity to regions within 50-200 bases of anintron-exon splice junction. It has been observed that some exonsequences can be included in the construct without seriously affectingthe target selectivity thereof. The amount of exonic material includedwill vary depending on the particular exon and intron sequences used.One can readily test whether too much exon DNA is included simply bytesting the constructs in vitro to determine whether normal cellularfunction is affected or whether the expression of related genes havingcomplementary sequences is affected.

As stated above, "complementary" or "antisense" means polynucleotidesequences that are substantially complementary over their entire lengthand have very few base mismatches. For example, sequences of fifteenbases in length may be termed complementary when they have complementarynucleotides at thirteen or fourteen positions. Naturally, sequenceswhich are completely complementary will be sequences which are entirelycomplementary throughout their entire length and have no basemismatches. Other sequences with lower degrees of homology also arecontemplated. For example, an antisense construct which has limitedregions of high homology, but also contains a non-homologous region(e.g., ribozyme; see below) could be designed. These molecules, thoughhaving less than 50% homology, would bind to target sequences underappropriate conditions.

It may be advantageous to combine portions of genomic DNA with cDNA orsynthetic sequences to generate specific constructs. For example, wherean intron is desired in the ultimate construct, a genomic clone willneed to be used. The cDNA or a synthesized polynucleotide may providemore convenient restriction sites for the remaining portion of theconstruct and, therefore, would be used for the rest of the sequence.

D. Ribozymes

Another approach for reducing APE expression is through the use ofribozymes. Although proteins traditionally have been used for catalysisof nucleic acids, another class of macromolecules has emerged as usefulin this endeavor. Ribozymes are RNA-protein complexes that cleavenucleic acids in a site-specific fashion. Ribozymes have specificcatalytic domains that possess endonuclease activity (Kim and Cech,1987; Gerlach et al., 1987; Forster and Symons, 1987). For exarnple, alarge number of ribozymes accelerate phosphoester transfer reactionswith a high degree of specificity, often cleaving only one of severalphosphoesters in an oligonucleotide substrate (Cech et al., 1981; Micheland Westhof, 1990; Reinhold-Hurek and Shub, 1992). This specificity hasbeen attributed to the requirement that the substrate bind via specificbase-pairing interactions to the internal guide sequence ("IGS") of theribozyme prior to chemical reaction.

Ribozyme catalysis has primarily been observed as part ofsequence-specific cleavage/ligation reactions involving nucleic acids(Joyce, 1989; Cech et al., 1981). For example, U.S. Pat. No. 5,354,855reports that certain ribozymes can act as endonucleases with a sequencespecificity greater than that of known ribonucleases and approachingthat of the DNA restriction enzymes. Thus, sequence-specificribozyme-mediated inhibition of gene expression may be particularlysuited to therapeutic applications (Scanlon et al., 1991; Sarver et al,1990). Recently, it was reported that ribozymes elicited genetic changesin some cells lines to which they were applied; the altered genesincluded the oncogenes H-ras, c-fos and genes of HIV. Most of this workinvolved the modification of a target mRNA, based on a specific mutantcodon that is cleaved by a specific ribozyme.

E. Vectors for Cloning, Gene Transfer and Expression

DNAs (coding, antisense, ribozymes) of the invention can be incorporatedinto viral, plasmid or other vectors and used to transform variousmammalian, bacterial or other cell types to achieve expression of theAPE protein in the cells. Illustrative mammalian cells include HeLa,endothelial, fibroblast, germ, brain, lung, liver, spleen, kidney, lymphnode, small intestine, pancreas, blood cells, colon, stomach, breast,endometrium, cervix, prostate, testicle, ovary, skin, head and neck,esophagus, bone marrow and blood tumor cells.

Expression requires that appropriate signals be provided in the vectors,and which include various regulatory elements, such asenhancers/promoters from both viral and mammalian sources that driveexpression of the genes of interest in host cells. Elements designed tooptimize messenger RNA stability and translatability in host cells alsoare defined. The conditions for the use of a number of dominant drugselection markers for establishing permanent, stable cell clonesexpressing the products are also provided, as is an element that linksexpression of the drug selection markers to expression of thepolypeptide.

(i) Regulatory Elements

Throughout this application, the term "expression construct" is meant toinclude any type of genetic construct containing a nucleic acid codingfor a gene product in which part or all of the nucleic acid encodingsequence is capable of being transcribed. The transcript may betranslated into a protein, but it need not be. In certain embodiments,expression includes both transcription of a gene and translation of mRNAinto a gene product. In other embodiments, expression only includestranscription of the nucleic acid encoding a gene of interest.

In preferred embodiments, the nucleic acid encoding a gene product isunder transcriptional control of a promoter. A "promoter" refers to aDNA sequence recognized by the synthetic machinery of the cell, orintroduced synthetic machinery, required to initiate the specifictranscription of a gene. The phrase "under transcriptional control"means that the promoter is in the correct location and orientation inrelation to the nucleic acid to control RNA polymerase initiation andexpression of the gene.

The term promoter will be used here to refer to a group oftranscriptional control modules that are clustered around the initiationsite for RNA polymerase II. Much of the thinking about how promoters areorganized derives from analyses of several viral promoters, includingthose for the HSV thymidine kinase (tk) and SV40 early transcriptionunits. These studies, augmented by more recent work, have shown thatpromoters are composed of discrete functional modules, each consistingof approximately 7-20 bp of DNA, and containing one or more recognitionsites for transcriptional activator or repressor proteins.

At least one module in each promoter functions to position the startsite for RNA synthesis. The best known example of this is the TATA box,but in some promoters lacking a TATA box, such as the promoter for themammalian terminal deoxynucleotidyl transferase gene and the promoterfor the SV40 late genes, a discrete element overlying the start siteitself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptionalinitiation. Typically, these are located in the region 30-110 bpupstream of the start site, although a number of promoters have recentlybeen shown to contain functional elements downstream of the start siteas well. The spacing between promoter elements frequently is flexible,so that promoter function is preserved when elements are inverted ormoved relative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either co-operatively or independently to activatetranscription.

The particular promoter employed to control the expression of a nucleicacid sequence of interest is not believed to be important, so long as itis capable of direction the expression of the nucleic acid in thetargeted cell. Thus, where a human cell is targeted, it is preferable toposition the nucleic acid coding region adjacent to and under thecontrol of a promoter that is capable of being expressed in a humancell. Generally speaking, such a promoter might include either a humanor viral promoter.

In various embodiments, the human cytomegalovirus (CMV) immediate earlygene promoter, the SV40 early promoter, the Rous sarcoma virus longterminal repeat, rat insulin promoter and glyceraldehyde-3-phosphatedehydrogenase, can be used to obtain high-level expression of the codingsequence of interest. In a preferred embodiment, retroviral LTRpromoters are employed in conjunction with the present invention. Theuse of other viral or mammalian cellular or bacterial phage promoterswhich are well-known in the art to achieve expression of a codingsequence of interest is contemplated as well, provided that the levelsof expression are sufficient for a given purpose.

By employing a promoter with well-known properties, the level andpattern of expression of the protein of interest following transfectionor transformation can be optimized. Further, selection of a promoterthat is regulated in response to specific physiologic signals can permitinducible expression of the gene product. Tables 2 and 3 list severalelements/promoters which may be employed, in the context of the presentinvention, to regulate the expression of the gene of interest. This listis not intended to be exhaustive of all the possible elements involvedin the promotion of gene expression but, merely, to be exemplarythereof.

Enhancers are genetic elements that increase transcription from apromoter located at a distant position on the same molecule of DNA.Enhancers are organized much like promoters. That is, they are composedof many individual elements, each of which binds to one or moretranscriptional proteins.

The basic distinction between enhancers and promoters is operational. Anenhancer region as a whole must be able to stimulate transcription at adistance; this need not be true of a promoter region or its componentelements. On the other hand, a promoter must have one or more elementsthat direct initiation of RNA synthesis at a particular site and in aparticular orientation, whereas enhancers lack these specificities.Promoters and enhancers are often overlapping and contiguous, oftenseeming to have a very similar modular organization.

Below is a list of viral promoters, cellular promoters/enhancers andinducible promoters/enhancers that could be used in combination with thenucleic acid encoding a gene of interest in an expression construct(Table 2 and Table 3). Additionally, any promoter/enhancer combination(as per the Eukaryotic Promoter Data Base EPDB) could also be used todrive expression of the gene. Eukaryotic cells can support cytoplasmictranscription from certain bacterial promoters if the appropriatebacterial polymerase is provided, either as part of the delivery complexor as an additional genetic expression construct.

                  TABLE 2    ______________________________________    ENHANCER/PROMOTER    ______________________________________    Immunoglobulin Heavy Chain    Immunoglobulin Light Chain    T-Cell Receptor    HLA DQ α and DQ β    β-Interferon    Interleukin-2    Interleukin-2 Receptor    MHC Class II 5    MHC Class II HLA-DRα    β-Actin    Muscle Creatine Kinase    Prealbumin (Transthyretin)    Elastase I    Metallothionein    Collagenase    Albumin Gene    α-Fetoprotein    τ-Globin    β-Globin    e-fos    c-HA-ras    Insulin    Neural Cell Adhesion Molecule (NCAM)    α1-Antitrypsin    H2B (TH2B) Histone    Mouse or Type I Collagen    Glucose-Regulated Proteins (GRP94 and GRP78)    Rat Growth Hormone    Human Serum Amyloid A (SAA)    Troponin I (TN I)    Platelet-Derived Growth Factor    Duchenne Muscular Dystrophy    SV40    Polyoma    Retroviruses    Papilloma Virus    Hepatitis B Virus    Human Immunodeficiency Virus    Cytomegalovirus    Gibbon APE Leukemia Virus    ______________________________________

                  TABLE 3    ______________________________________    Element          Inducer    ______________________________________    MT II            Phorbol Ester (TPA)                     Heavy metals    MMTV (mouse mammary tumor                     Glucocorticoids    virus)    β-Interferon                     poly (rI) X                     poly (rc)    Adenovirus 5 E2  Ela    c-jun            Phorbol Ester (TPA), H.sub.2 O.sub.2    Collagenase      Phorbol Ester (TPA)    Stromelysin      Phorbol Ester (TPA), IL-1    SV40             Phorbol Ester (TPA)    Murine MX Gene   Interferon, Newcastle Disease Virus    GRP78 Gene       A23187    α-2-Macroglobulin                     IL-6    Vimentin         Serum    MHC Class I Gene H-2kB                     Interferon    HSP70            Ela, SV40 Large T Antigen    Proliferin       Phorbol Ester-TPA    Tumor Necrosis Factor                     FMA    Thyroid Stimulating Hormone α                     Thyroid Hormone    Gene    Insulin E Box    Glucose    ______________________________________

Where a cDNA insert is employed, one will typically desire to include apolyadenylation signal to effect proper polyadenylation of the genetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed such as human growth hormone and SV40polyadenylation signals. Also contemplated as an element of theexpression cassette is a terminator. These elements can serve to enhancemessage levels and to minimize read through from the cassette into othersequences.

(ii) Selectable Markers

In certain embodiments of the invention, the cells contain nucleic acidconstructs of the present invention, a cell may be identified in vitroor in vivo by including a marker in the expression construct. Suchmarkers would confer an identifiable change to the cell permitting easyidentification of cells containing the expression construct. Usually theinclusion of a drug selection marker aids in cloning and in theselection of transformants, for example, genes that confer resistance toneomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol areuseful selectable markers. Alternatively, enzymes such as herpes simplexvirus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT)may be employed. Immunologic markers also can be employed. Theselectable marker employed is not believed to be important, so long asit is capable of being expressed simultaneously with the nucleic acidencoding a gene product. Further examples of selectable markers are wellknown to one of skill in the art.

(iii) Multigene Constructs and IRES

In certain embodiments of the invention, the use of internal ribosomebinding sites (IRES) elements are used to create multigene, orpolycistronic, messages. IRES elements are able to bypass the ribosomescanning model of 5' methylated Cap dependent translation and begintranslation at internal sites (Pelletier and Sonenberg, 1988). IRESelements from two members of the picanovirus family (polio andencephalomyocarditis) have been described (Pelletier and Sonenberg,1988), as well an IRES from a mammalian message (Macejak and Sarnow,1991). IRES elements can be linked to heterologous open reading frames.Multiple open reading frames can be transcribed together, each separatedby an IRES, creating polycistronic messages. By virtue of the IRESelement, each open reading frame is accessible to ribosomes forefficient translation. Multiple genes can be efficiently expressed usinga single promoter/enhancer to transcribe a single message.

Any heterologous open reading frame can be linked to IRES elements. Thisincludes genes for secreted proteins, multi-subunit proteins encoded byindependent genes, intracellular or membrane-bound proteins andselectable markers. In this way, expression of several proteins can besimultaneously engineered into a cell with a single construct and asingle selectable marker.

(iv) Delivery of Expression Vectors

There are a number of ways in which expression vectors may introducedinto cells. In certain embodiments of the invention, the expressionconstruct comprises a virus or engineered construct derived from a viralgenome. The ability of certain viruses to enter cells viareceptor-mediated endocytosis, to integrate into host cell genome andexpress viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign genes into mammalian cells(Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden,1986; Temin, 1986). The first viruses used as gene vectors were DNAviruses including the papovaviruses (simian virus 40, bovine papillomavirus, and polyoma) (kidgeway, 1988; Baichwal and Sugden, 1986) andadenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986). These have arelatively low capacity for foreign DNA sequences and have a restrictedhost spectrum. Furthermore, their oncogenic potential and cytopathiceffects in permissive cells raise safety concerns. They can accommodateonly up to 8 kB of foreign genetic material but can be readilyintroduced in a variety of cell lines and laboratory animals (Nicolasand Rubenstein, 1988; Temin, 1986).

One of the preferred methods for in vivo delivery involves the use of anadenovirus expression vector. "Adenovirus expression vector" is meant toinclude those constructs containing adenovirus sequences sufficient to(a) support packaging of the construct and (b) to express an antisensepolynucleotide that has been cloned therein. In this context, expressiondoes not require that the gene product be synthesized.

The expression vector comprises a genetically engineered form ofadenovirus. Knowledge of the genetic organization of adenovirus, a 36kB, linear, double-stranded DNA virus, allows substitution of largepieces of adenoviral DNA with foreign sequences up to 7 kB (Grunhaus andHorwitz, 1992). In contrast to retrovirus, the adenoviral infection ofhost cells does not result in chromosomal integration because adenoviralDNA can replicate in an episomal manner without potential genotoxicity.Also, adenoviruses are structurally stable, and no genome rearrangementhas been detected after extensive amplification. Adenovirus can infectvirtually all epithelial cells regardless of their cell cycle stage. Sofar, adenoviral infection appears to be linked only to mild disease suchas acute respiratory disease in humans.

Adenovirus is particularly suitable for use as a gene transfer vectorbecause of its mid-sized genome, ease of manipulation, high titer, widetarget cell range and high infectivity. Both ends of the viral genomecontain 100-200 base pair inverted repeats (ITRs), which are ciselements necessary for viral DNA replication and packaging. The early(E) and late (L) regions of the genome contain different transcriptionunits that are divided by the onset of viral DNA replication. The E1region (E1A and E1B) encodes proteins responsible for the regulation oftranscription of the viral genome and a few cellular genes. Theexpression of the E2 region (E2A and E2B) results in the synthesis ofthe proteins for viral DNA replication. These proteins are involved inDNA replication, late gene expression and host cell shut-off (Renan,1990). The products of the late genes, including the majority of theviral capsid proteins, are expressed only after significant processingof a single primary transcript issued by the major late promoter (MLP).The MLP, (located at 16.8 m.u.) is particularly efficient during thelate phase of infection, and all the mRNA's issued from this promoterpossess a 5'-tripartite leader (TPL) sequence which makes them preferredmRNA's for translation.

In a current system, recombinant adenovirus is generated from homologousrecombination between shuttle vector and provirus vector. Due to thepossible recombination between two proviral vectors, wild-typeadenovirus may be generated from this process. Therefore, it is criticalto isolate a single clone of virus from an individual plaque and examineits genomic structure.

Generation and propagation of the current adenovirus vectors, which arereplication deficient, depend on a unique helper cell line, designated293, which was transformed from human embryonic kidney cells by Ad5 DNAfragments and constitutively expresses E1 proteins (Graham et al, 1977).Since the E3 region is dispensable from the adenovirus genome (Jones andShenk, 1978), the current adenovirus vectors, with the help of 293cells, carry foreign DNA in either the E1, the D3 or both regions(Graham and Prevec, 1991). In nature, adenovirus can packageapproximately 105% of the wild-type genome (Ghosh-Choudhury et al.,1987), providing capacity for about 2 extra kB of DNA. Combined with theapproximately 5.5 kB of DNA that is replaceable in the E1 and E3regions, the maximum capacity of the current adenovirus vector is under7.5 kB, or about 15% of the total length of the vector. More than 80% ofthe adenovirus viral genome remains in the vector backbone and is thesource of vector-borne cytotoxicity. Also, the replication deficiency ofthe E1-deleted virus is incomplete. For example, leakage of viral geneexpression has been observed with the currently available vectors athigh multiplicities of infection (MOI) (Mulligan, 1993).

Helper cell lines may be derived from human cells such as humanembryonic kidney cells, muscle cells, hematopoietic cells or other humanembryonic mesenchymal or epithelial cells. Alternatively, the helpercells may be derived from the cells of other mammalian species that arepermissive for human adenovirus. Such cells include, e.g., Vero cells orother monkey embryonic mesenchymal or epithelial cells. As stated above,the preferred helper cell line is 293.

Recently, Racher et al., (1995) disclosed improved methods for culturing293 cells and propagating adenovirus. In one format, natural cellaggregates are grown by inoculating individual cells into 1 litersiliconized spinner flasks (Techne, Cambridge, UK) containing 100-200 mlof medium. Following stirring at 40 rpm, the cell viability is estimatedwith trypan blue. In another format, Fibra-Cel microcarriers (BibbySterlin, Stone, UK) (5 g/l) is employed as follows. A cell inoculum,resuspended in 5 ml of medium, is added to the carrier (50 ml) in a 250ml Erlenmeyer flask and left stationary, with occasional agitation, for1 to 4 h. The medium is then replaced with 50 ml of fresh medium andshaking initiated. For virus production, cells are allowed to grow toabout 80% confluence, after which time the medium is replaced (to 25% ofthe final volume) and adenovirus added at an MOI of 0.05. Cultures areleft stationary overnight, following which the volume is increased to100% and shaking commenced for another 72 h.

Other than the requirement that the adenovirus vector be replicationdefective, or at least conditionally defective, the nature of theadenovirus vector is not believed to be crucial to the successfulpractice of the invention. The adenovirus may be of any of the 42different known serotypes or subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain theconditional replication-defective adenovirus vector for use in thepresent invention. This is because Adenovirus type 5 is a humanadenovirus about which a great deal of biochemical and geneticinformation is known, and it has historically been used for mostconstructions employing adenovirus as a vector.

As stated above, the typical vector according to the present inventionis replication defective and will not have an adenovirus E1 region.Thus, it will be most convenient to introduce the polynucleotideencoding the gene of interest at the position from which the E1-codingsequences have been removed. However, the position of insertion of theconstruct within the adenovirus sequences is not critical to theinvention. The polynucleotide encoding the gene of interest may also beinserted in lieu of the deleted E3 region in E3 replacement vectors asdescribed by Karlsson et al., (1986) or in the E4 region where a helpercell line or helper virus complements the E4 defect.

Adenovirus is easy to grow and manipulate and exhibits broad host rangein vitro and in vivo. This group of viruses can be obtained in hightiters, e.g., 10₉ -10₁₁ plaque-forming units per ml, and they are highlyinfective. The life cycle of adenovirus does not require integrationinto the host cell genome. The foreign genes delivered by adenovirusvectors are episomal and, therefore, have low genotoxicity to hostcells. No side effects have been reported in studies of vaccination withwild-type adenovirus (Couch et al., 1963; Top et al., 1971),demonstrating their safety and therapeutic potential as in vivo genetransfer vectors.

Adenovirus vectors have been used in eukaryotic gene expression (Levreroet al, 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhausand Horwitz, 1992; Graham and Prevec, 1992). Recently, animal studiessuggested that recombinant adenovirus could be used for gene therapy(Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet etal., 1990; Rich et al., 1993). Studies in administering recombinantadenovirus to different tissues include trachea instillation (Rosenfeldet al, 1991; Rosenfeld et al., 1992), muscle injection (Ragot et al.,1993), peripheral intravenous injections (Herz and Gerard, 1993) andstereotactic inoculation into the brain (Le Gal La Salle et al., 1993).

In a particularly preferred embodiment, the present invention employsretroviral vectors for delivery. The retroviruses are a group ofsingle-stranded RNA viruses characterized by an ability to convert theirRNA to double-stranded DNA in infected cells by a process ofreverse-transcription (Coffin, 1990). The resulting DNA then stablyintegrates into cellular chromosomes as a provirus and directs synthesisof viral proteins. The integration results in the retention of the viralgene sequences in the recipient cell and its descendants. The retroviralgenome contains three genes, gag, pol, and env that code for capsidproteins, polymerase enzyme, and envelope components, respectively. Asequence found upstream from the gag gene contains a signal forpackaging of the genome into virions. Two long terminal repeat (LTR)sequences are present at the 5' and 3' ends of the viral genome. Thesecontain strong promoter and enhancer sequences and are also required forintegration in the host cell genome (Coffin, 1990).

In order to construct a retroviral vector, a nucleic acid encoding agene of interest is inserted into the viral genome in the place ofcertain viral sequences to produce a virus that isreplication-defective. In order to produce virions, a packaging cellline containing the gag, pol, and env genes but without the LTR andpackaging components is constructed (Mann et al., 1983). When arecombinant plasmid containing a cDNA, together with the retroviral LTRand packaging sequences is introduced into this cell line (by calciumphosphate precipitation for example), the packaging sequence allows theRNA transcript of the recombinant plasmid to be packaged into viralparticles, which are then secreted into the culture media (Nicolas andRubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containingthe recombinant retroviruses is then collected, optionally concentrated,and used for gene transfer. Retroviral vectors are able to infect abroad variety of cell types. However, integration and stable expressionrequire the division of host cells (Paskind et al., 1975).

A novel approach designed to allow specific targeting of retrovirusvectors was recently developed based on the chemical modification of aretrovirus by the chemical addition of lactose residues to the viralenvelope. This modification could permit the specific infection ofhepatocytes via sialoglycoprotein receptors.

A different approach to targeting of recombinant retroviruses wasdesigned in which biotinylated antibodies against a retroviral envelopeprotein and against a specific cell receptor were used. The antibodieswere coupled via the biotin components by using streptavidin (Roux etal., 1989). Using antibodies against major histocompatibility complexclass I and class II antigens, they demonstrated the infection of avariety of human cells that bore those surface antigens with anecotropic virus in vitro Roux et al, 1989).

There are certain limitations to the use of retrovirus vectors in allaspects of the present invention. For example, retrovirus vectorsusually integrate into random sites in the cell genome. This can lead toinsertional mutagenesis through the interruption of host genes orthrough the insertion of viral regulatory sequences that can interferewith the flnction of flanking genes (Varmus et al, 1981). Anotherconcern with the use of defective retrovirus vectors is the potentialappearance of wild-type replication-competent virus in the packagingcells. This can result from recombination events in which the intact-sequence from the recombinant virus inserts upstream from the gag, pol,env sequence integrated in the host cell genome. However, new packagingcell lines are now available that should greatly decrease the likelihoodof recombination (Markowitz et al., 1988; Hersdorffer et al, 1990). Inparticular, the present inventor employs retroviral plasmid transfectedinto the packaging lines GP+E86 and GP+AM12 (Markowitz et al, 1988a;Markowitz et al., 1988b). The helper virus genome of the packaging lineis separated onto two plasmids. The packaging signal and 3' LTR havebeen removed. Construction of these retroviral lines make the generationof recombinant retrovirus uulikely since at least three recombinationalevents must occur prior to generation of wild type virus.

Other viral vectors may be employed as expression constructs in thepresent invention. Vectors derived from viruses such as vaccinia virus(Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988)adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986;Hermonat and Muzycska, 1984) and herpesviruses may be employed. Theyoffer several attractive features for various mammalian cells(Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar etal, 1988; Horwich et al, 1990).

With the recent recognition of defective hepatitis B viruses, newinsight was gained into the structure-function relationship of differentviral sequences. In vitro studies showed that the virus could retain theability for helper-dependent packaging and reverse transcription despitethe deletion of up to 80% of its genome (Horwich et al., 1990). Thissuggested that large portions of the genome could be replaced withforeign genetic material. The hepatotropism and persistence(integration) were particularly attractive properties for liver-directedgene transfer. Chang et al, recently introduced the chloramphenicolacetyltransferase (CAT) gene into duck hepatitis B virus genome in theplace of the polymerase, surface, and pre-surface coding sequences. Itwas co-transfected with wild-type virus into an avian hepatoma cellline. Culture media containing high titers of the recombinant virus wereused to infect primary duckling hepatocytes. Stable CAT gene expressionwas detected for at least 24 days after transfection (Chang et al.,1991).

In order to effect expression of sense or antisense gene constructs, theexpression construct must be delivered into a cell. This delivery may beaccomplished in vitro, as in laboratory procedures for transformingcells lines, or in vivo or ex vivo, as in the treatment of certaindisease states. One mechanism for delivery is via viral infection wherethe expression construct is encapsidated in an infectious viralparticle.

Several non-viral methods for the transfer of expression constructs intocultured mammalian cells also are contemplated by the present invention.These include calcium phosphate precipitation (Graham and Van Der Eb,1973; Chen and Okayama, 1987; Rippe et al, 1990) DEAE-dextran (Gopal,1985), electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984),direct microinjection (Harland and Weintraub, 1985), DNA-loadedliposomes (Nicolau and Sene, 1982; Fraley et al., 1979) andlipofectamine-DNA complexes, cell sonication (Fechheimer et al., 1987),gene bombardment using high velocity microprojectiles (Yang et al,1990), and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu,1988). Some of these techniques may be successfully adapted for in vivoor ex vivo use.

Once the expression construct has been delivered into the cell thenucleic acid encoding the gene of interest may be positioned andexpressed at different sites. In certain embodiments, the nucleic acidencoding the gene may be stably integrated into the genome of the cell.This integration may be in the cognate location and orientation viahomologous recombination (gene replacement) or it may be integrated in arandom, non-specific location (gene augmentation). In yet ftrtherembodiments, the nucleic acid may be stably maintained in the cell as aseparate, episomal segment of DNA. Such nucleic acid segments or"episomes" encode sequences sufficient to permit maintenance andreplication independent of or in synchronization with the host cellcycle. How the expression construct is delivered to a cell and where inthe cell the nucleic acid remains is dependent on the type of expressionconstruct employed.

In yet another embodiment of the invention, the expression construct maysimply consist of naked recombinant DNA or plasmids. Transfer of theconstruct may be performed by any of the methods mentioned above whichphysically or chemically permeabilize the cell membrane. This isparticularly applicable for transfer in vitro but it may be applied toin vivo use as well. Dubensky et al. (1984) successfully injectedpolyomavirus DNA in the form of calcium phosphate precipitates intoliver and spleen of adult and newborn mice demonstrating active viralreplication and acute infection. Benvenisty and Neshif (1986) alsodemonstrated that direct intraperitoneal injection of calciumphosphate-precipitated plasmids results in expression of the transfectedgenes. It is envisioned that DNA encoding a gene of interest may also betransferred in a similar manner in vivo and express the gene product.

In still another embodiment, the transferring a naked DNA expressionconstruct into cells may involve particle bombardment. This methoddepends on the ability to accelerate DNA-coated microprojectiles to ahigh velocity allowing them to pierce cell membranes and enter cellswithout killing them (Klein et al, 1987). Several devices foraccelerating small particles have been developed. One such device relieson a high voltage discharge to generate an electrical current, which inturn provides the motive force (Yang et al, 1990). The microprojectilesused have consisted of biologically inert substances such as tungsten orgold beads.

Selected organs including the liver, skin, and muscle tissue of rats andmice have been bombarded in vivo (Yang et al., 1990; Zelenin et al.,1991). This may require surgical exposure of the tissue or cells, toeliminate any intervening tissue between the gun and the target organ,ie., ex vivo treatment. Again, DNA encoding a particular gene may bedelivered via this method and still be incorporated by the presentinvention.

In a further embodiment of the invention, the expression construct maybe entrapped in a liposome. Liposomes are vesicular structurescharacterized by a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). Also contemplated are lipofectamine-DNA complexes.

Liposome-mediated nucleic acid delivery and expression of foreign DNA invitro has been very successful. Wong et al., (1980) demonstrated thefeasibility of liposome-mediated delivery and expression of foreign DNAin cultured chick embryo, HeLa and hepatoma cells. Nicolau et al.,(1987) accomplished successful liposome-mediated gene transfer in ratsafter intravenous injection.

In certain embodiments of the invention, the liposome may be complexedwith a hemagglutinating virus (HVJ). This has been shown to facilitatefusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments,the liposome may be complexed or employed in conjunction with nuclearnon-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yetfurther embodiments, the liposome may be complexed or employed inconjunction with both HVJ and HMG-1. In that such expression constructshave been successfully employed in transfer and expression of nucleicacid in vitro and in vivo, then they are applicable for the presentinvention. Where a bacterial promoter is employed in the DNA construct,it also will be desirable to include within the liposome an appropriatebacterial polymerase.

Other expression constructs which can be employed to deliver a nucleicacid encoding a particular gene into cells are receptor-mediateddelivery vehicles. These take advantage of the selective uptake ofmacromolecules by receptor-mediated endocytosis in almost all eukaryoticcells. Because of the cell type-specific distribution of variousreceptors, the delivery can be highly specific (Wu and Wu, 1993).

Receptor-mediated gene targeting vehicles generally consist of twocomponents: a cell receptor-specific ligand and a DNA-binding agent.Several ligands have been used for receptor-mediated gene transfer. Themost extensively characterized ligands are asialoorosomucoid (ASOR) (Wuand Wu, 1987) and transferring (Wagner et al., 1990). Recently, asynthetic neoglycoprotein, which recognizes the same receptor as ASOR,has been used as a gene delivery vehicle (Ferkol et al., 1993; Peraleset al., 1994) and epidermal growth factor (EGF) has also been used todeliver genes to squamous carcinoma cells (Myers, EPO 0273085).

In other embodiments, the delivery vehicle may comprise a ligand and aliposome. For example, Nicolau et al., (1987) employedlactosyl-ceramide, a galactose-terminal asialganglioside, incorporatedinto liposomes and observed an increase in the uptake of the insulingene by hepatocytes. Thus, it is feasible that a nucleic acid encoding aparticular gene also may be specifically delivered into a cell type suchas lung, epithelial or tumor cells, by any number of receptor-ligandsystems with or without liposomes. For example, epidermal growth factor(EGF) may be used as the receptor for mediated delivery of a nucleicacid encoding a gene in many tumor cells that exhibit upregulation ofEGF receptor. Mannose can be used to target the mannose receptor onliver cells. Also, antibodies to CD5 (CLL), CD22 (lymphoma), CD25(T-cell leukemia) and MAA (melanoma) can similarly be used as targetingmoieties.

Primary mammalian cell cultures may be prepared in various ways. Inorder for the cells to be kept viable while in vitro and in contact withthe expression construct, it is necessary to ensure that the cellsmaintain contact with the correct ratio of oxygen and carbon dioxide andnutrients but are protected from microbial contamination. Cell culturetechniques are well documented and are disclosed herein by reference(Freshner, 1992).

(v) Use of genes to transform host cells

One embodiment of the foregoing involves the use of gene transfer toinunortalize cells for the production of proteins. The gene for theprotein of interest may be transferred as described above intoappropriate host cells followed by culture of cells under theappropriate conditions. The gene for virtually any polypeptide may beemployed in this manner. The generation of recombinant expressionvectors, and the elements included therein, are discussed above.Alternatively, the protein to be produced may be an endogenous proteinnormally synthesized by the cell in question.

Examples of useful mammalian host cell lines are Vero and HeLa cells andcell lines of Chinese hamster ovary, W138, BHK, COS-7, 293, HepG2,NIH3T3, RIN and MDCK cells. In addition, a host cell strain may bechosen that modulates the expression of the inserted sequences, ormodifies and process the gene product in the manner desired. Suchmodifications (e.g., glycosylation) and processing (e.g., cleavage) ofprotein products may be important for the function of the protein.Different host cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins. Appropriatecell lines or host systems can be chosen to insure the correctmodification and processing of the foreign protein expressed.

A number of selection systems may be used including, but not limited to,HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase andadenine phosphoribosyltransferase genes, in tk-, hgprt- or aprt- cells,respectively. Also, antimetabolite resistance can be used as the basisof selection for dhfr, that confers resistance to; gpt, that confersresistance to mycophenolic acid; neo, that confers resistance to theaminoglycoside G418; and hygro, that confers resistance to hygromycin.

Animal cells can be propagated in vitro in two modes: as non-anchoragedependent cells growing in suspension throughout the bulk of the cultureor as anchorage-dependent cells requiring attachment to a solidsubstrate for their propagation (i.e., a monolayer type of cell growth).

Non-anchorage dependent or suspension cultures from continuousestablished cell lines are the most widely used means of large scaleproduction of cells and cell products. However, suspension culturedcells have limitations, such as tumorigenic potential and lower proteinproduction than adherent T-cells.

Large scale suspension culture of mammalian cells in stirred tanks is acommon method for production of recombinant proteins. Two suspensionculture reactor designs are in wide use--the stirred reactor and theairlift reactor. The stirred design has successfully been used on an8000 liter capacity for the production of interferon. Cells are grown ina stainless steel tank with a height-to-diameter ratio of 1:1 to 3:1.The culture is usually mixed with one or more agitators, based on bladeddisks or marine propeller patterns. Agitator systems offering less shearforces than blades have been described. Agitation may be driven eitherdirectly or indirectly by magnetically coupled drives. Indirect drivesreduce the risk of microbial contamination through seals on stirrershafts.

The airlift reactor, also initially described for microbial fermentationand later adapted for mammalian culture, relies on a gas stream to bothmix and oxygenate the culture. The gas stream enters a riser section ofthe reactor and drives circulation. Gas disengages at the culturesurface, causing denser liquid free of gas bubbles to travel downward inthe downcomer section of the reactor. The main advantage of this designis the simplicity and lack of need for mechanical mixing. Typically, theheight-to-diameter ratio is 10:1. The airlift reactor scales uprelatively easily, has good mass transfer of gases and generatesrelatively low shear forces.

IV. GENERATING ANTIBODIES REACTIVE WITH THE APE PROTEIN

As stated above, an important aspect of the present inventioncontemplates production of antibodies that are immunoreactive with anAPE protein molecule of the present invention, or any portion thereof.An antibody can be a polyclonal or a monoclonal antibody. In a preferredembodiment, an antibody is a monoclonal antibody. Means for preparingand characterizing antibodies are well known in the art (see, e.g.,Harlow and Lane, 1988).

Briefly, a polyclonal antibody is prepared by immunizing an animal withan immunogen comprising a polypeptide of the present invention andcollecting antisera from that immunized animal. A wide range of animalspecies can be used for the production of antisera. Typically an animalused for production of anti-antisera is a non-human animal includingchickens, rabbits, mice, rats, hamsters, pigs or horses. Because of therelatively large blood volume of rabbits, a rabbit is a preferred choicefor production of polyclonal antibodies.

Antibodies, both polyclonal and monoclonal, specific for isoforms ofantigen may be prepared using conventional immunization techniques, aswill be generally known to those of skill in the art. A compositioncontaining antigenic epitopes of the compounds of the present inventioncan be used to immunize one or more experimental animals, such as arabbit or mouse, which will then proceed to produce specific antibodiesagainst the compounds of the present invention. Polyclonal antisera maybe obtained, after allowing time for antibody generation, simply bybleeding the animal and preparing serum samples from the whole blood.

It is proposed that the monoclonal antibodies of the present inventionwill find useful application in standard immunochemical procedures, suchas ELISA and Western blot methods and in immunohistochemical proceduressuch as tissue staining, as well as in other procedures which mayutilize antibodies specific to APE-related antigen epitopes.Additionally, it is proposed that monoclonal antibodies specific to theparticular constructs may be utilized in other useful applications.

In general, both polyclonal and monoclonal antibodies against APEconstructs of the present invention may be used in a variety ofembodiments. For example, they may be employed in antibody cloningprotocols to obtain cDNAs or genes encoding other APE construct. Theymay also be used in inhibition studies to analyze the effects of APEconstruct related peptides in cells or animals. Anti-APE constructantibodies will also be useful in immunolocalization studies to analyzethe distribution of APE during various cellular events, for example, todetermine the cellular or tissue-specific distribution of APEpolypeptides under different points in the cell cycle. A particularlyuseful application of such antibodies is in purifying native orrecombinant APE, for example, using an antibody affinity column. Theoperation of all such immunological techniques will be known to those ofskill in the art in light of the present disclosure.

Means for preparing and characterizing antibodies are well known in theart (see, e.g., Harlow and Lane, 1988; incorporated herein byreference). More specific examples of monoclonal antibody preparationare give in the examples below.

As is well known in the art, a given composition may vary in itsimnuunogenicity. It is often necessary therefore to boost the hostinunune system, as may be achieved by coupling a peptide or polypeptideimnmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin can alsobe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

As also is well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Exemplary andpreferred adjuvants include complete Freund's adjuvant (a non-specificstimulator of the immune response containing killed Mycobacteriumtuberculosis), incomplete Freund's adjuvants and aluminum hydroxideadjuvant.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen (subcutaneous, intramuscular, intradermal, intravenous andintraperitoneal). The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization. A second, booster, injection may also be given.The process of boosting and titering is repeated until a suitable titeris achieved. When a desired level of immunogenicity is obtained, theimmunized animal can be bled and the serum isolated and stored, and/orthe animal can be used to generate mAbs.

MAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, e.g., a purified orpartially purified APE protein, polypeptide or peptide or cellexpressing high levels of APE. The immunizing composition isadministered in a manner effective to stimulate antibody producingcells. Rodents such as mice and rats are preferred animals, however, theuse of rabbit, sheep frog cells is also possible. The use of rats mayprovide certain advantages (Goding, 1986), but mice are preferred, withthe BALB/c mouse being most preferred as this is most routinely used andgenerally gives a higher percentage of stable fusions.

Following immunization, somatic cells with the potential for producingantibodies, specifically B-lymphocytes (B-cells), are selected for usein the mAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible. Often, a panel of animals will have beenimmunized and the spleen of animal with the highest antibody titer willbe removed and the spleen lymphocytes obtained by homogenizing thespleen with a syringe. Typically, a spleen from an immunized mousecontains approximately 5×10⁷ to 2×10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, 1986; Campbell, 1984). For example, wherethe immunized animal is a mouse, one may use P3-X63/Ag8, P3-X63-Ag8.653,NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC 11-X45-GTG 1.7 andS194/5XX0Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all usefulin connection with cell fusions.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1to about 1:1, respectively, in the presence of an agent or agents(chemical or electrical) that promote the fusion of cell membranes.Fusion methods using Sendai virus have been described (Kohler andMilstein, 1975; 1976), and those using polyethylene glycol (PEG), suchas 37% (v/v) PEG, by Gefter et al., (1977). The use of electricallyinduced fusion methods is also appropriate (Goding, 1986).

Fusion procedures usually produce viable hybrids at low frequencies,around 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B-cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B-cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide mAbs. The cell lines may be exploitedfor mAb production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,can then be tapped to provide mAbs in high concentration. The individualcell lines could also be cultured in vitro, where the mAbs are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations. mAbs produced by either means may be furtherpurified, if desired, using filtration, centrifugation and variouschromatographic methods such as HPLC or affinity chromatography.

V. DIAGNOSTIC APPLICATIONS.

According to the present invention, the level of APE increases in cellsthat are in a (pre)malignant state. This stands in direct contrast toother repair enzymes, such as MGMT, MAG, ERCC1, MDR-1, DNA topoisomeraseI, DNA topoisomerase IIα a and GSTπ. Codegoni et al. (1997). Thus, APErepresents an indicator of cancers such as cervical, brain, lung, liver,spleen, kidney, lymph node, small intestine, pancreas, blood cells,colon, stomach, breast, endometrium, prostate, cervix, testicle, ovary,skin, head and neck, esophagus, bone marrow and blood tumor cells. Thusmonitoring levels of APE will provide an indication of the whether thecell is a normal cell or a (pre)malignant cell, allowing early detectingand intervention in (pre)malignant conditions.

In other aspects, the present invention shows that the level of APEdeclines as cells undergo apoptosis. Thus, APE represents an earlyindicator of apoptotic activity. There are a variety of reasons onewould seek to determine the apoptotic state of a cell. In a preferredembodiment, this information will provide a clinician with feed back onthe efficacy of treatment designed to induce apoptosis, e.g., chemo-,radio- or gene therapy for cancer. For example, if a given treatmentdoes not result in a drop in APE levels by a predetermined time, it maybe in the patient's interest to cease the treatment. Similarly, iflevels of APE drop in response to a given therapy, the apoptotic actionof the therapy may be facilitated and no more therapy is needed. In boththese scenarios, undesired toxic effects of the therapy may be avoidedwithout loss of benefit.

In another context, it also may be desirable to examine the apoptoticstate of a target cell to determine whether normal or abnormal cellaging, senescence and/or death is occurring. For example, the reductionof certain T-cell populations in immunodeficiency diseases may involveinduction of apoptotic functions which could be identified, monitoredand treated according to the present invention. For example, in a recentstudy it was suggested that HIV-1 infected individuals display multiplesymptoms of redox imbalance consistent with oxidative stress and theirlymphocytes are much more prone to undergo apoptosis in vitro. Oxidativestress is a physiological mediator of programmed cell death inlymphocytes and so HIV is an extreme example of what can happen whenregulatory safeguards are compromised. Thus the present invention couldbe used to monitor and treat such individuals.

Thus, it will be desirable examine APE protein levels, APE transcriptionand at the APE structural gene and regulatory regions. The biologicalsample can be any tissue or fluid. Various embodiments include cells ofthe skin, muscle, facia, brain, prostate, breast, endometrium, lung,head & neck, pancreas, small intestine, blood cells, liver, testes,ovaries, colon, skin, stomach, esophagus, spleen, lymph node, bonemarrow or kidney. Other embodiments include fluid samples such asperipheral blood, lymph fluid, ascites, serous fluid, pleural effusion,sputum, cerebrospinal fluid, lacrimal fluid, stool or urine.

A. Genetic Diagnosis

One embodiment of the instant invention comprises a method for detectingan increase in the expression of APE by looking at the APE transcriptsof a cell or the copy number of the gene. Another embodiment of theinstant invention comprises a method for detecting reduction in theexpression or function of APE by examining at the genes and transcriptsof a cell. Nucleic acid used is isolated from cells contained in thebiological sample, according to standard methodologies (Sambrook et al.,1989). The nucleic acid may be genomic DNA or fractionated or whole cellRNA. Where RNA is used, it may be desired to convert the RNA to acomplementary DNA. In one embodiment, the RNA is whole cell RNA; inanother, it is poly-A RNA. Normally, the nucleic acid is amplified.

Depending on the format, the specific nucleic acid of interest isidentified in the sample directly using amplification or with a second,known nucleic acid following amplification. Next, the identified productis detected. In certain applications, the detection may be performed byvisual means (e.g., ethidium bromide staining of a gel). Alternatively,the detection may involve indirect identification of the product viachemiluminescence, radioactive scintigraphy of radiolabel or fluorescentlabel or even via a system using electrical or thermal impulse signals(Affymax Technology; Bellus, 1994).

Following detection, one may compare the results seen in a given patientwith a control reaction or a statistically significant reference groupof normal patients. In this way, it is possible to correlate the amountof APE detected with apoptotic states.

In addition to determining levels of APE, it also may prove useful toexamine various types of defects. These defect could include deletions,insertions, point mutations and duplications. Point mutations result instop codons, frameshift mutations or amino acid substitutions. Somaticmutations are those occurring in non-germline tissues. Germ-line tissuecan occur in any tissue and are inherited. Mutations in and outside thecoding region also may affect the amount of APE produced, both byaltering the transcription of the gene or in destabilizing or otherwisealtering the processing of either the transcript (mRNA) or protein.

A variety of different assays are contemplated in this regard, includingbut not limited to, fluorescent in situ hybridization (FISH), direct DNAsequencing, PFGE analysis, Southern or Northern blotting,single-stranded conformation analysis (SSCA), RNAse protection assay,allele-specific oligonucleotide (ASO), dot blot analysis, denaturinggradient gel electrophoresis, RFLP and PCR-SSCP.

(i) Primers and Probes

The term primer, as defmed herein, is meant to encompass any nucleicacid that is capable of priming the synthesis of a nascent nucleic acidin a template-dependent process. Typically, primers are oligonucleotidesfrom ten to twenty base pairs in length, but longer sequences can beemployed. Primers may be provided in double-stranded or single-strandedform, although the single-stranded form is preferred. Probes are defineddifferently, although they may act as primers. Probes, while perhapscapable of priming, are designed to binding to the target DNA or RNA andneed not be used in an amplification process.

In preferred embodiments, the probes or primers are labeled withradioactive species (³² P, ¹⁴ C, ³⁵ S, ³ H, or other label), with afluorophore (rhodamine, fluorescein) or a chemillumiscent (luciferase).

(ii) Template Dependent Amplification Methods

A number of template dependent processes are available to amplify themarker sequences present in a given template sample. One of the bestknown amplification methods is the polymerase chain reaction (referredto as PCR™) which is described in detail in U.S. Pat. Nos. 4,683,195,4,683,202 and 4,800,159, and in Innis et al., 1990, each of which isincorporated herein by reference in its entirety.

Briefly, in PCR, two primer sequences are prepared that arecomplementary to regions on opposite complementary strands of the markersequence. An excess of deoxynucleoside triphosphates are added to areaction mixture along with a DNA polymerase, e.g., Taq polymerase. Ifthe marker sequence is present in a sample, the primers will bind to themarker and the polymerase will cause the primers to be extended alongthe marker sequence by adding on nucleotides. By raising and loweringthe temperature of the reaction mixture, the extended primers willdissociate from the marker to form reaction products, excess primerswill bind to the marker and to the reaction products and the process isrepeated.

A reverse transcriptase PCR amplification procedure may be performed inorder to quantify the amount of mRNA amplified. Methods of reversetranscribing RNA into cDNA are well known and described in Sambrook etal., 1989. Alternative methods for reverse transcription utilizethermostable, RNA-dependent DNA polymerases. These methods are describedin WO 90/07641 filed Dec. 21, 1990. Polymerase chain reactionmethodologies are well known in the art.

Another method for amplification is the ligase chain reaction ("LCR"),disclosed in EPO No. 320 308, incorporated herein by reference in itsentirety. In LCR, two complementary probe pairs are prepared, and in thepresence of the target sequence, each pair will bind to oppositecomplementary strands of the target such that they abut. In the presenceof a ligase, the two probe pairs will link to form a single unit. Bytemperature cycling, as in PCR, bound ligated units dissociate from thetarget and then serve as "target sequences" for ligation of excess probepairs. U.S. Pat. No. 4,883,750 describes a method similar to LCR forbinding probe pairs to a target sequence.

Qbeta Replicase, described in PCT Application No. PCT/US87/00880, mayalso be used as still another amplification method in the presentinvention. In this method, a replicative sequence of RNA that has aregion complementary to that of a target is added to a sample in thepresence of an RNA polymerase. The polymerase will copy the replicativesequence that can then be detected.

An isothermal amplification method, in which restriction endonucleasesand ligases are used to achieve the amplification of target moleculesthat contain nucleotide 5'- alpha-thio!-triphosphates in one strand of arestriction site may also be useful in the amplification of nucleicacids in the present invention, Walker et al., (1992).

Strand Displacement Amplification (SDA) is another method of carryingout isothermal amplification of nucleic acids which involves multiplerounds of strand displacement and synthesis, i.e., nick translation. Asimilar method, called Repair Chain Reaction (RCR), involves annealingseveral probes throughout a region targeted for amplification, followedby a repair reaction in which only two of the four bases are present.The other two bases can be added as biotinylated derivatives for easydetection. A similar approach is used in SDA. Target specific sequencescan also be detected using a cyclic probe reaction (CPR). In CPR, aprobe having 3' and 5' sequences of non-specific DNA and a middlesequence of specific RNA is hybridized to DNA that is present in asample. Upon hybridization, the reaction is treated with RNase H, andthe products of the probe identified as distinctive products that arereleased after digestion. The original template is annealed to anothercycling probe and the reaction is repeated.

Still another amplification methods described in GB Application No. 2202 328, and in PCT Application No. PCT/US89/01025, each of which isincorporated herein by reference in its entirety, may be used inaccordance with the present invention. In the former application,"modified" primers are used in a PCR-like, template- andenzyme-dependent synthesis. The primers may be modified by labeling witha capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme).In the latter application, an excess of labeled probes are added to asample. In the presence of the target sequence, the probe binds and iscleaved catalytically. After cleavage, the target sequence is releasedintact to be bound by excess probe. Cleavage of the labeled probesignals the presence of the target sequence.

Other nucleic acid amplification procedures include transcription-basedamplification systems (TAS), including nucleic acid sequence basedamplification (NASBA) and 3SR (Kwoh et al., 1989; Gingeras et al, PCTApplication WO 88/10315, incorporated herein by reference in theirentirety). In NASBA, the nucleic acids can be prepared for amplificationby standard phenol/chloroform extraction, heat denaturation of aclinical sample, treatment with lysis buffer and minispin columns forisolation of DNA and RNA or guanidinium chloride extraction of RNA.These amplification techniques involve annealing a primer which hastarget specific sequences. Following polymerization, DNA/RNA hybrids aredigested with RNase H while double stranded DNA molecules are heatdenatured again. In either case the single stranded DNA is made fullydouble stranded by addition of second target specific primer, followedby polymerization. The double-stranded DNA molecules are then multiplytranscribed by an RNA polymerase such as T7 or SP6. In an isothermalcyclic reaction, the RNA's are reverse transcribed into single strandedDNA, which is then converted to double stranded DNA, and thentranscribed once again with an RNA polymerase such as T7 or SP6. Theresulting products, whether truncated or complete, indicate targetspecific sequences.

Davey et al., EPO No. 329 822 (incorporated herein by reference in itsentirety) disclose a nucleic acid amplification process involvingcyclically synthesizing single-stranded RNA ("ssRNA"), ssDNA, anddouble-stranded DNA (dsDNA), which may be used in accordance with thepresent invention. The ssRNA is a template for a first primeroligonucleotide, which is elongated by reverse transcriptase(RNA-dependent DNA polymerase). The RNA is then removed from theresulting DNA:RNA duplex by the action of ribonuclease H (RNase H, anRNase specific for RNA in duplex with either DNA or RNA). The resultantssDNA is a template for a second primer, which also includes thesequences of an RNA polymerase promoter (exemplified by T7 RNApolymerase) 5' to its homology to the template. This primer is thenextended by DNA polymerase (exemplified by the large "Klenow" fragmentof E. coli DNA polymerase I), resulting in a double-stranded DNA("dsDNA") molecule, having a sequence identical to that of the originalRNA between the primers and having additionally, at one end, a promotersequence. This promoter sequence can be used by the appropriate RNApolymerase to make many RNA copies of the DNA. These copies can thenre-enter the cycle leading to very swift amplification. With properchoice of enzymes, this amplification can be done isothermally withoutaddition of enzymes at each cycle. Because of the cyclical nature ofthis process, the starting sequence can be chosen to be in the form ofeither DNA or RNA.

Miller et al., PCT Application WO 89/06700 (incorporated herein byreference in its entirety) disclose a nucleic acid sequenceamplification scheme based on the hybridization of a promoter/primersequence to a target single-stranded DNA ("ssDNA") followed bytranscription of many RNA copies of the sequence. This scheme is notcyclic, i.e., new templates are not produced from the resultant RNAtranscripts. Other amplification methods include "RACE" and "one-sidedPCR" (Frohman, M. A., In: PCR PROTOCOLS: A GUIDE TO METHODS,ANDAPPLICATIONS, Academic Press, N.Y., 1990; Ohara et al., 1989; eachherein incorporated by reference in their entirety).

Methods based on ligation of two (or more) oligonucleotides in thepresence of nucleic acid having the sequence of the resulting"di-oligonucleotide", thereby amplifying the di-oligonucleotide, mayalso be used in the amplification step of the present invention. Wu etal., (1989), incorporated herein by reference in its entirety.

(iii) Southern/Northern Blotting

Blotting techniques are well known to those of skill in the art.Southern blotting involves the use of DNA as a target, whereas Northernblotting involves the use of RNA as a target. Each provide differenttypes of information, although cDNA blotting is analogous, in manyaspects, to blotting or RNA species.

Briefly, a probe is used to target a DNA or RNA species that has beenimmobilized on a suitable matrix, often a filter of nitrocellulose. Thedifferent species should be spatially separated to facilitate analysis.This often is accomplished by gel electrophoresis of nucleic acidspecies followed by "blotting" on to the filter.

Subsequently, the blotted target is incubated with a probe (usuallylabeled) under conditions that promote denaturation and rehybridization.Because the probe is designed to base pair with the target, the probewill binding a portion of the target sequence under renaturingconditions. Unbound probe is then removed, and detection is accomplishedas described above.

(iv) Separation Methods

It normally is desirable, at one stage or another, to separate theamplification product from the template and the excess primer for thepurpose of determining whether specific amplification has occurred. Inone embodiment, amplification products are separated by agarose,agarose-acrylamide or polyacrylamide gel electrophoresis using standardmethods. See Sambrook et al., 1989.

Alternatively, chromatographic techniques may be employed to effectseparation. There are many kinds of chromatography which may be used inthe present invention: adsorption, partition, ion-exchange and molecularsieve, and many specialized techniques for using them including column,paper, thin-layer and gas chromatography (Freifelder, 1982).

(v) Detection Methods

Products may be visualized in order to confirm amplification of themarker sequences. One typical visualization method involves staining ofa gel with ethidium bromide and visualization under UV light.Alternatively, if the amplification products are integrally labeled withradio- or fluorometrically-labeled nucleotides, the amplificationproducts can then be exposed to x-ray film or visualized under theappropriate stimulating spectra, following separation.

In one embodiment, visualization is achieved indirectly. Followingseparation of amplification products, a labeled nucleic acid probe isbrought into contact with the amplified marker sequence. The probepreferably is conjugated to a chromophore but may be radiolabeled. Inanother embodiment, the probe is conjugated to a binding partner, suchas an antibody or biotin, and the other member of the binding paircarries a detectable moiety.

In one embodiment, detection is by a labeled probe. The techniquesinvolved are well known to those of skill in the art and can be found inmany standard books on molecular protocols. See Sambrook et al., 1989.For example, chromophore or radiolabel probes or primers identify thetarget during or following amplification.

One example of the foregoing is described in U.S. Pat. No. 5,279,721,incorporated by reference herein, which discloses an apparatus andmethod for the automated electrophoresis and transfer of nucleic acids.The apparatus permits electrophoresis and blotting without externalmanipulation of the gel and is ideally suited to carrying out methodsaccording to the present invention.

In addition, the amplification products described above may be subjectedto sequence analysis to identify specific kinds of variations usingstandard sequence analysis techniques. Within certain methods,exhaustive analysis of genes is carried out by sequence analysis usingprimer sets designed for optimal sequencing (Pignon et al, 1994). Thepresent invention provides methods by which any or all of these types ofanalyses may be used. Using the sequences disclosed herein,oligonucleotide primers may be designed to permit the amplification ofsequences throughout the APE gene construct that may then be analyzed bydirect sequencing.

(vi) Kit Components

All the essential materials and reagents required for detecting andsequencing APE and variants thereof may be assembled together in a kit.This generally will comprise preselected primers and probes. Alsoincluded may be enzymes suitable for amplifying nucleic acids includingvarious polymerases (RT, Taq, Sequenase™ etc.), deoxynucleotides andbuffers to provide the necessary reaction mixture for amplification.Such kits also generally will comprise, in suitable means, distinctcontainers for each individual reagent and enzyme as well as for eachprimer or probe.

(vii) Design and Theoretical Considerations for Relative QuantitativeRT-PCR

Reverse transcription (RT) of RNA to cDNA followed by relativequantitative PCR (RT-PCR) can be used to determine the relativeconcentrations of specific mRNA species isolated from patients. Bydetermining that the concentration of a specific mRNA species varies, itis shown that the gene encoding the specific mRNA species isdifferentially expressed.

In PCR, the number of molecules of the amplified target DNA increase bya factor approaching two with every cycle of the reaction until somereagent becomes linmiting. Thereafter, the rate of amplification becomesincreasingly diminished until there is no increase in the amplifiedtarget between cycles. If a graph is plotted in which the cycle numberis on the X axis and the log of the concentration of the amplifiedtarget DNA is on the Y axis, a curved line of characteristic shape isformed by connecting the plotted points. Beginning with the first cycle,the slope of the line is positive and constant. This is said to be thelinear portion of the curve. After a reagent becomes limiting, the slopeof the line begins to decrease and eventually becomes zero. At thispoint the concentration of the amplified target DNA becomes asymptoticto some fixed value. This is said to be the plateau portion of thecurve.

The concentration of the target DNA in the linear portion of the PCRamplification is directly proportional to the starting concentration ofthe target before the reaction began. By determining the concentrationof the amplified products of the target DNA in PCR reactions that havecompleted the same number of cycles and are in their linear ranges, itis possible to determine the relative concentrations of the specifictarget sequence in the original DNA mixture. If the DNA mixtures arecDNAs synthesized from RNAs isolated from different tissues or cells,the relative abundances of the specific mRNA from which the targetsequence was derived can be determined for the respective tissues orcells. This direct proportionality between the concentration of the PCRproducts and the relative mRNA abundances is only true in the linearrange of the PCR reaction.

The final concentration of the target DNA in the plateau portion of thecurve is determined by the availability of reagents in the reaction mixand is independent of the original concentration of target DNA.Therefore, the first condition that must be met before the relativeabundances of a mRNA species can be determined by RT-PCR for acollection of RNA populations is that the concentrations of theamplified PCR products must be sampled when the PCR reactions are in thelinear portion of their curves.

The second condition that must be met for an RT-PCR experiment tosuccessfiully determine the relative abundances of a particular mRNAspecies is that relative concentrations of the amplifiable cDNAs must benormalized to some independent standard. The goal of an RT-PCRexperiment is to determine the abundance of a particular mRNA speciesrelative to the average abundance of all mRNA species in the sample. Inthe experiments described below, mRNAs for β-actin, asparaginesynthetase and lipocortin II were used as external and internalstandards to which the relative abundance of other mRNAs are compared.

Most protocols for competitive PCR utilize internal PCR standards thatare approximately as abundant as the target. These strategies areeffective if the products of the PCR amplifications are sampled duringtheir linear phases. If the products are sampled when the reactions areapproaching the plateau phase, then the less abundant product becomesrelatively over represented. Comparisons of relative abundances made formany different RNA samples, such as is the case when examining RNAsamples for differential expression, become distorted in such a way asto make differences in relative abundances of RNAs appear less than theyactually are. This is not a significant problem if the internal standardis much more abundant than the target. If the internal standard is moreabundant than the target, then direct linear comparisons can be madebetween RNA samples.

The above discussion describes theoretical considerations for an RT-PCRassay for clinically derived materials. The problems inherent inclinical samples are that they are of variable quantity (makingnormalization problematic), and that they are of variable quality(necessitating the co-amplification of a reliable internal control,preferably of larger size than the target). Both of these problems areovercome if the RT-PCR is performed as a relative quantitative RT-PCRwith an internal standard in which the internal standard is anamplifiable cDNA fragment that is larger than the target cDNA fragmentand in which the abundance of the mRNA encoding the internal standard isroughly 5-100 fold higher than the mRNA encoding the target. This assaymeasures relative abundance, not absolute abundance of the respectivemRNA species.

Other studies may be performed using a more conventional relativequantitative RT-PCR assay with an external standard protocol. Theseassays sample the PCR products in the linear portion of theiramplification curves. The number of PCR cycles that are optimal forsampling must be empirically determined for each target cDNA fragment.In addition, the reverse transcriptase products of each RNA populationisolated from the various tissue samples must be carefully normalizedfor equal concentrations of amplifiable cDNAs. This consideration isvery important since the assay measures absolute mRNA abundance.Absolute mRNA abundance can be used as a measure of differential geneexpression only in normalized samples. While empirical determination ofthe linear range of the amplification curve and normalization of cDNApreparations are tedious and time consuming processes, the resultingRT-PCR assays can be superior to those derived from the relativequantitative RT-PCR assay with an internal standard.

One reason for this advantage is that without the internalstandard/competitor, all of the reagents can be converted into a singlePCR product in the linear range of the amplification curve, thusincreasing the sensitivity of the assay. Another reason is that withonly one PCR product, display of the product on an electrophoretic gelor another display method becomes less complex, has less background andis easier to interpret.

(viii) Chip Technologies

Specifically contemplated by the present inventor are chip-based DNAtechnologies such as those described by Hacia et al. (1996) andShoemaker et al. (1996). Briefly, these techniques involve quantitativemethods for analyzing large numbers of genes rapidly and accurately. Bytagging genes with oligonucleotides or using fixed probe arrays, one canemploy chip technology to segregate target molecules as high densityarrays and screen these molecules on the basis of hybridization. Seealso Pease et al. (1994); Fodor et al. (1991).

B. Immunodiagnosis

Antibodies of the present invention can be used in measuring an increaseor decrease in APE expression in healthy and diseased tissues, throughtechniques such as ELISAs and Western blotting. Illustrative assaystrategies which can be used to detect APE include, but are not limitedto, immunoassays involving the binding of antibodies (polyclonal ormonoclonal) to APE in the sample, and the analysis of the sample forbound antibodies. A number of human and other mammalian antibodies toAPE are known, and are available either commercially or throughtechniques well known to the art and industry. Another suitable antibodymaterial can be obtained, for instance, by raising rabbit antibodiesagainst recombinantly-derived APE as generally described in Example 1below and in (Duguid et al., 1995). Moreover, for this and other aspectsof the invention, it will be understood that the APE antibody structurecan be genetically manipulated or incorporated in fusion proteinswithout departing from the invention. Accordingly, antibodies can beused in the present invention in their natural or genetically alteredforms.

The use of antibodies of the present invention, in an ELISA assay iscontemplated. For example, anti-APE antibodies are immobilized onto aselected surface, preferably a surface exhibiting a protein affinitysuch as the wells of a polystyrene microtiter plate. After washing toremove incompletely adsorbed material, it is desirable to bind or coatthe assay plate wells with a non-specific protein that is known to beantigenically neutral with regard to the test antisera such as bovineserum albumin (BSA), casein or solutions of powdered milk. This allowsfor blocking of non-specific adsorption sites on the immobilizingsurface and thus reduces the background caused by non-specific bindingof antigen onto the surface.

After binding of antibody to the well, coating with a non-reactivematerial to reduce background, and washing to remove unbound material,the immobilizing surface is contacted with the sample to be tested in amanner conducive to immune complex (antigen/antibody) formation.

Following formation of specific immunocomplexes between the test sampleand the bound antibody, and subsequent washing, the occurrence and evenamount of immunocomplex formation may be determined by subjecting sameto a second antibody having specificity for APE that differs the firstantibody. Appropriate conditions preferably include diluting the samplewith diluents such as BSA, bovine gamma globulin (BGG) and phosphatebuffered saline (PBS)/Tween®. These added agents also tend to assist inthe reduction of nonspecific background. The layered antisera is thenallowed to incubate for from about 2 to about 4 hr, at temperaturespreferably on the order of about 25° to about 27° C. Followingincubation, the antisera-contacted surface is washed so as to removenon-immunocomplexed material. A preferred washing procedure includeswashing with a solution such as PBS/Tween®, or borate buffer.

To provide a detecting means, the second antibody will preferably havean associated enzyme that will generate a color development uponincubating with an appropriate chromogenic substrate. Thus, for example,one will desire to contact and incubate the second antibody-boundsurface with a urease or peroxidase-conjugated anti-human IgG for aperiod of time and under conditions which favor the development ofimmunocomplex formation (e.g., incubation for 2 hr at room temperaturein a PBS-containing solution such as PBS/Tween®).

After incubation with the second enzyme-tagged antibody, and subsequentto washing to remove unbound material, the amount of label is quantifiedby incubation with a chromogenic substrate such as urea and bromocresolpurple or 2,2'-azino-di-(3-ethyl-benzthiazoline)-6-sulfonic acid (ABTS)and H₂ O₂, in the case of peroxidase as the enzyme label. Quantitationis then achieved by measuring the degree of color generation, e.g.,using a visible spectrum spectrophotometer.

The preceding format may be altered by first binding the sample to theassay plate. Then, primary antibody is incubated with the assay plate,followed by detecting of bound primary antibody using a labeled secondantibody with specificity for the primary antibody.

The antibody compositions of the present invention will find great usein immunoblot or Western blot analysis. The antibodies may be used ashigh-affinity primary reagents for the identification of proteinsimmobilized onto a solid support matrix, such as nitrocellulose, nylonor combinations thereof. In conjunction with immunoprecipitation,followed by gel electrophoresis, these may be used as a single stepreagent for use in detecting antigens against which secondary reagentsused in the detection of the antigen cause an adverse background.Immunologically-based detection methods for use in conjunction withWestern blotting include enzymatically-, radiolabel-, orfluorescently-tagged secondary antibodies against the toxin moiety areconsidered to be of particular use in this regard.

Detection of bound antibodies to APE can be accomplished in any suitablefashion, including for example the use of labeled APE antibody which iscontacted with the sample under binding conditions. In addition, thesample can be contacted with APE antibody under binding conditions, andthen subsequently contacted with a label which specifically binds to APEantibody, e.g., a labeled antigen. In this regard, suitable labels willinclude chemiluminescent, fluorescent, or radionuclear compounds,microparticles, enzymes, or other labels which are known to be usefulfor detection.

Another detection strategy which can be used in the invention includes amicrosphere agglutination assay (MAA). In such assays, uniformly sizedspheres of plastic (e.g., of 30 nanometer to 1 micron in diameter,preferably about 100 to 400 nanometers in diameter) may have finctionalgroups on their surfaces for covalent attachment of biomolecules. Whilethe use of functional groups is common, it is not always necessary asmany biomolecules will adhere to plastic itself. As an example, onepartner of a specific binding pair (i.e., antibody or antigen) isattached to the surface of the microspheres. The other partner is placedinto solution with the microspheres to initiate the assay, and will actas a bridge between microspheres so as to cause agglutination or"clumping" of the microspheres. The rate of agglutination is directlydependent upon the concentration of the binding pair partner insolution. The level of agglutination can be assayed by light scatteringor light absorbence of the sample, wherein the amount of light whichpasses through the solution is measured either at one point in time orat several points in time to derive a rate of change in the amount oflight which gets through. This provides an accurate measure of the levelof agglutination of the microspheres since they scatter more light thansingle (non-agglutinated) microspheres. This measure than then becorrelated to the concentration of binding partner in the solution.

Thus, in one strategy, the inventive methods can employ an MAA in whichthe specific binding pair is an APE antibody (Ab) and APE. In aso-called direct assay, the antibody is bound to the surface of themicroparticles, so as to enable the assay of a sample for the amount ofAPE it contains. The APE sample, which can for instance include apreparation containing contents of a cellular specimen, is added to themicrospheres, and the level of APE is determined by light scatteringmeasurements generally as described above.

In an MAA inhibition assay, APE levels may be measured indirectly. Forthe assay, APE is attached to the surface of the microparticles, and APEAb is diluted into a second reagent buffer at a fixed concentration. Thesample to be assayed for APE level is added to the Ab reagent, so thatany APE in the sample binds to the Ab. The microsphere reagent is addedto begin the reaction, causing agglutination. The rate of agglutinationis thus directly proportional to the amount of Ab and inverselyproportional to the amount of APE in the sample.

It will be well understood that other means of testing APE levels areavailable, including, for instance, those involving testing for analtered level of APE enzymatic activity, or Western blot analysis of APEprotein levels in tissues or cells using APE antibody, or assaying theamount of antibody or other APE binding partner which is not bound to asample, and subtracting from the total amount of antibody or bindingpartner added.

VI. METHODS FOR SCREENING ACTIVE COMPOUNDS

The present invention also contemplates the use of APE and activefragments, nucleic acids coding therefor, and recombinant cellsexpressing APE at low and high levels in the screening of compounds forinhibition of APE activity or reducing APE expression. Such compoundswould be important in a number of aspects. They would be important inregimens for the treatment of APE-related cancers, whether administeredalone or in combination with chemo- and radiotherapeutic regimens in thetreatment of cancer. Alternatively, by simply reducing APE, thesecompounds will be instrumental in initiating programmed cell death. Inother instances, it may be desirable to determine if a compound iscapable of increasing APE levels, thereby preventing apoptosis in cellswhich have abnormally low APE activity.

These assays may make use of a variety of different formats and maydepend on the kind of "activity" for which the screen is beingconducted. Contemplated finctional "read-outs" include alterations inAPE expression levels, binding to APE or an APE cofactor, inhibition ofAPE binding to a substrate, apoptosis, presence or lack of growth,presence or lack of metastasis, presence or lack of cell division,presence or lack of cell migration, presence or lack of soft agar colonyformation, presence or lack of contact inhibition, presence or lack ofinvasiveness, or presence or lack of tumor progression or othermalignant phenotype.

In addition to testing of single compounds, it may be useful to testcombinations of different compounds, especially known compounds such aschemotherapeutic agents. Other combinations might include a compoundplus u.v. or ionizing (alpha, beta or gamma) radiation.

A. Cell-Free Assays

In one embodiment, the invention is to be applied to the screening ofcompounds that bind to the APE molecule or fragment thereof. Thepolypeptide or fragment may be either free in solution, fixed to asupport, expressed in or on the surface of a cell. Either thepolypeptide or the compound may be labeled, thereby permitting adetermination of binding.

In another embodiment, the assay may measure the inhibition of bindingof APE to a natural or artificial substrate or binding partner.Competitive binding assays can be performed in which one of the agents(APE, binding partner or compound) is labeled. Usually, the polypeptidewill be the labeled species. One may measure the amount of free labelversus bound label to determine binding or inhibition of binding.

Another technique for high-throughput screening of compounds isdescribed in WO 84/03564. Large numbers of small peptide test compoundsare synthesized on a solid substrate, such as plastic pins or some othersurface. The peptide test compounds are reacted with APE and washed.Bound polypeptide is detected by various methods.

Purified APE can be coated directly onto plates for use in theaforementioned drug screening techniques. However, non-neutralizingantibodies to the polypeptide can be used to immobilize the polypeptideto a solid phase. Also, fusion proteins containing a reactive region(preferably a terminal region) may be used to link the APE active regionto a solid phase.

Once identified in this format of assay, it is likely that additionalstudies designed to elucidate the finctional effects of the agent on APEwill be conducted, for example, as described below.

B. Cell-Based Assays

Various cell lines containing wild-type or natural or engineeredmutations in APE can be used to study various functional attributes ofAPE and how a candidate compound affects these attributes. Methods forengineering genetic constructs are described elsewhere in this document.In such assays, the compound would be formulated appropriately, givenits biochemical nature, and contacted with a target cell. Depending onthe assay, culture may be required. The cell may then be examined byvirtue of a number of different physiologic assays. These might includemeasurement of cell growth, division, contact inhibition, metastasis,soft agar formation or other characteristic. Alternatively, molecularanalysis may be performed in which the molecular function or state ofAPE, or related pathways, may be explored. This may involve assays suchas those for protein expression, enzyme function, substrate utilization,phosphorylation states of various molecules including APE, cAMP levels,mRNA expression (including differential display of whole cell or polyARNA) and others described above.

In particular embodiments, the present invention concerns a method foridentifying compounds that will modulate expression of wild-type APE.Useful compounds in this regard will not be limited to those mentionedin the present application. The active compounds may include fragmentsor parts of naturally-occurring compounds or may be only found as activecombinations of known compounds which are otherwise inactive. However,prior to testing of such compounds in humans or animal models, it may benecessary to test a variety of candidates to determine which havepotential.

Accordingly, in screening assays to identify pharmaceutical agents whichmodulate APE expression in cells, it is proposed that compounds isolatedfrom natural sources, such as animals, bacteria, fungi, plant sources,including leaves and bark, and marine samples may be assayed ascandidates for the presence of potentially useful pharmaceutical agents.It will be understood that the pharmaceutical agents to be screenedcould also be derived or synthesized from chemical compositions orman-made compounds.

In these embodiments, the present invention is directed to a method fordetermining the ability of a candidate substance to decrease thewild-type APE expression of cells and to concomitantly induce apoptosisin said cells, the method including generally the steps of:

(a) obtaining a cell with wild-type APE;

(b) admixing a candidate substance with the cell; and

(c) determining the ability of the candidate substance to reduce the APEcontent of the cell.

To identify a candidate substance as being capable of modulating APEexpression, one would measure or determine the APE status of a cell. Ifthat cell has the ability to express APE, its basal APE content in theabsence of the added candidate substance is measured. One would then addthe candidate substance to the cell and redetermine the wild-type APE inthe presence of the candidate substance. A candidate substance whichdecreases the APE expression relative to the cell's APE expression inthe absence of the substance is indicative of a candidate substance withwild-type APE expression inhibiting capability, and will therefor havetherapeutic cancer reducing and apoptotic potential as described in thepresent invention.

Conversely it may be useful to increase the APE level in cells that areundergoing premature cell death or to halt apoptosis, in this regard itwill be useful to employ a screening assay that will identify candidatesubstances that increase or enhance the expression and activity of APE.This screening assay is quite similar to that described above to measureincreases in decreases in APE levels. After obtaining an suitable testcell, one will admix a candidate substance with the cell a measure APElevels to determine if increases in APE synthesis or steady state levelshave occurred.

"Effective amounts", in certain circumstances, are those amountseffective at reproducibly decreasing APE expression in cells incomparison to their normal levels. Compounds that achieve significantappropriate changes in activity will be used. If desired, a battery ofcompounds may be screened in vitro to identify other agents for use inthe present invention.

C. In Vivo Assays

The present invention also encompasses the use of various animal models.By developing or isolating cell lines that express APE at altered levelsor express APE variants, one can generate disease models in variouslaboratory animals. These models may employ the orthotopic or systemicadministration of cells to mimic various disease states. Alternatively,one may APE-related disease states in animals by providing agents knownto affect APE levels. Finally, transgenic animals that lack oroverexpress a wild-type APE or an altered/mutant APE such as an an APEwith the redox region deleted or mutated, may be utilized as models fortreatment. Again, animal models provide a useful vehicle for testingcombinations of agents as well.

Treatment of animals with test compounds will involve the administrationof the compound, in an appropriate form, to the animal. Administrationwill be by any route the could be utilized for clinical or non-clinicalpurposes, including but not limited to oral, nasal, buccal, rectal,vaginal or topical. Alternatively, administration may be byintratracheal instillation, bronchial instillation, intradermal,subcutaneous, intramuscular, intraperitoneal or intravenous injection.Specifically contemplated are systemic intravenous injection, regionaladministration via blood or lymph supply and intratumnoral injection.

Determining the effectiveness of a compound in vivo may involve avariety of different criteria. Such criteria include, but are notlimited to, survival, reduction of tumor burden or mass, arrest orslowing of tumor progression, elimination of tumors, inhibition orprevention of metastasis, increased activity level, improvement inimmune effector function and improved food intake.

D. Rational Drug Design

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptides or compounds with which they interact(agonists, antagonists, inhibitors, binding partners, etc.). By creatingsuch analogs, it is possible to fashion drugs which are more active orstable than the natural molecules, which have different susceptibilityto alteration or which may affect the function of various othermolecules. In one approach, one would generate a three-dimensionalstructure for APE or a fragment thereof. This could be accomplished byx-ray crystallography, computer modeling or by a combination of bothapproaches. An alternative approach, "alanine scan," involves the randomreplacement of residues throughout molecule with alanine, and theresulting affect on function determined.

It also is possible to isolate a various proteins, selected by afunctional assay, and then solve its crystal structure. In principle,this approach yields a pharmacore upon which subsequent drug design canbe based. For antibodies, it is possible to bypass 1.-2 proteincrystallography by generating anti-idiotypic antibodies to a functional,pharmacologically active antibody. As a mirror image of a mirror image,the binding site of anti-idiotype would be expected to be an analog ofthe original antigen. The anti-idiotype could then be used to identifyand isolate peptides from banks of chemically- or biologically-producedpeptides. Selected peptides would then serve as the pharmacore.Anti-idiotypes may be generated using the methods described herein forproducing antibodies, using an antibody as the antigen. Anti-idiotypeapproaches may be applied, in theory, to any target binding protein.

VII. THERAPEUTIC APPLICATIONS

The present invention involves, in another embodiment, the treatment ofcancer. The types of cancer that may be treated, according to thepresent invention, is limited only by the involvement of APE. Byinvolvement, it is not even a requirement that APE be mutated orabnormal, but merely that an abnormally high level of APE expression bepresent. Thus, it is contemplated that a wide variety of tumors may betreated using APE therapy, including cancers of the cervix, prostate,brain (glioblastoma, astrocytoma, oligodendroglioma, ependymomas),liver, kidney, brain, breast, colon, stomach, head & neck, skin, bonemarrow, blood, lung, adenocarcinoma, and other tissues.

In many contexts, it is not necessary that the tumor cell be killed orinduced to undergo normal cell death or "apoptosis." Rather, toaccomplish a meaningful treatment, all that is required is that thetumor growth be slowed to some degree. It may be that the tumor growthis completely blocked, however, or that some tumor regression isachieved. Clinical terminology such as "remission" and "reduction oftumor" burden also are contemplated given their normal usage.

The present invention also involves the use of APE, APE genes, APEantibodies APE activity modulation and determination of APE levels intherapeutic settings. The methodologies for effecting these therapiesare set forth throughout the specification. The following is an outlineof how those of skill in the art would proceed in attaining thesetherapeutic goals.

A. Genetic Based Therapies

Thus, one of the therapeutic embodiments contemplated by the presentinvention is the intervention, at the molecular level, in the expressionof APE. The present inventor intends to provide to a cell an expressionconstruct capable of increasing or decreasing APE levels that cell. In aparticular embodiment, the present invention contempaltes providing to acell an expression construct capable of providing an antisense APE tosaid cell. Such a construct will be therapeutically effective as ananticancer agent, which results in a decrease, abrogation or eliminationof cancer cell growth or tumor size. In alternative embodiments, suchgene based therapies are provided to a cell to increase apoptosis insaid cell.

Any nucleic acid encoding an APE protein, as described herein, could beutilized, as could any of the sequence variants discussed above whichwould encode the same, or a biologically equivalent polypeptide.Different constraints are placed on the use of antisense constructs,which require specific levels of identity to achieve hybridization. Thelengthy discussion of expression vectors and the genetic elementsemployed therein is incorporated into this section by reference.Particularly preferred expression vectors are viral vectors such asadenovirus, adeno-associated virus, herpes virus, vaccinia virus andretrovirus. Also preferred is liposomally-encapsulated expressionvector.

Those of skill in the art are well aware of how to apply gene deliveryto in vivo and ex vivo situations. For viral vectors, one generally willprepare a viral vector stock. Depending on the kind of virus and thetiter attainable, one will deliver about 1×10⁴, 1 ×10⁵, 1×10⁶, 1×10⁷,1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹ or 1×10 ¹² infectious particles to thepatient. Similar figures may be extrapolated for liposomal or othernon-viral formulations by comparing relative uptake efficiencies.Formulation as a pharmaceutically acceptable composition is discussedbelow.

Various routes are contemplated for various tumor types. The sectionbelow on routes contains an extensive list of possible routes. Forpractically any setting, systemic delivery is contemplated. This willprove especially important, for example, in attacking microscopic ormetastatic cancer. Where a discrete target cell or tissue site may beidentified, a variety of direct, local and regional approaches may betaken. For example, an organ may be directly injected with theexpression vector. Also, a tumor bed may be treated prior to, during orafter resection. Following resection, one generally will deliver thevector by a catheter left in place following surgery. One may utilizethe appropriate surrounding vasculature to introduce the vector into thetumor by injecting a supporting vein or artery. A more distal bloodsupply route also may be utilized.

In a different embodiment, ex vivo gene therapy is contemplated. Thisapproach is particularly suited, although not limited, to treatment ofbone marrow associated cancers. In an ex vivo embodiment, cells from thepatient are removed and maintained outside the body for at least someperiod of time. During this period, a therapy is delivered, after whichthe cells are reintroduced into the patient.

Autologous bone marrow transplant (ABMT) is an example of ex vivo genetherapy. Basically, the notion behind ABMT is that the patient willserve as his or her own bone marrow donor. Thus, a normally lethal doseof irradiation or chemotherapeutic may be delivered to the patient tokill tumor cells, and the bone marrow repopulated with the patients owncells that have been maintained (and perhaps expanded) ex vivo. Because,bone marrow often is contaminated with tumor cells, it is desirable topurge the bone marrow of these cells. Use of gene therapy to accomplishthis goal is yet another way an anti-APE may be utilized according tothe present invention.

B. Augmenting Classic Chemo-, Radio- and Genetic Therapies

In another embodiment, the reduction of APE activity levels in tumorcells may augment the response of those cells to other kinds of cancertherapy. The mechanism by which this phenomenon occurs are not wellestablished, but given the role of APE in DNA repair, the loss of thisfunction may well lead to irreparable damage and, hence, apoptosis inaffected cells.

Reduction of APE activity may be achieved by one of a variety ofdifferent mechanisms. Clearly, if a single compound is available thatwill specifically reduce APE function, this would be the preferredoption. One example of such an agent is an antisense construct thatwould target APE genes and transcripts, thereby preventing transcriptionor processing/translation, respectively. Another approach might be theuse of antibodies, or corresponding single-chain antibody geneconstructs. The former suffers from constraints in importing the ratherlarge (160 Kd) molecule into the cell in a finctional state. Yet anotherapproach would be to provide a peptide or mimetic that would mimic partof the APE molecule such that the peptide or mimetic would effectivelycompete with APE but not perform APE-like function.

Agents or factors suitable for use in a combined therapy includeradiation and waves that induce DNA damage such as, γ-irradiation andX-rays and the like. Other forms of DNA damaging factors are alsocontemplated such as microwaves and UV-irradiation. It is most likelythat all of these factors effect a broad range of damage DNA, on theprecursors of DNA, the replication and repair of DNA, and the assemblyand maintenance of chromosomes. Dosage ranges for X-rays range fromdaily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4weeks), to single doses of 2000 to 6000 roentgens. Dosage ranges forradioisotopes vary widely, and depend on the half-life of the isotope,the strength and type of radiation emitted, and the uptake by theneoplastic cells.

A variety of chemical compounds, also described as "chemotherapeuticagents," function to induce DNA damage, all of which are intended to beof use in the combined treatment methods disclosed herein. For example,agents that directly cross-link nucleic acids, specifically DNA, areenvisaged to facilitate DNA damage leading to a synergistic,antineoplastic combination with APE modulation. Agents such ascisplatin, and other DNA alkylating agents may be used. Cisplatin hasbeen widely used to treat cancer, with efficacious doses used inclinical applications of 20 mg/m² for 5 days every three weeks for atotal of three courses. Cisplatin is not absorbed orally and musttherefore be delivered via injection intravenously, subcutaneously,intratumorally or intraperitoneally.

Agents that damage DNA also include compounds that interfere with DNAreplication, mitosis and chromosomal segregation. Such chemotherapeuticcompounds include adriamycin, also known as doxorubicin, etoposide,verapamil, podophyllotoxin, and the like. Widely used in a clinicalsetting for the treatment of neoplasms, these compounds are administeredthrough bolus injections intravenously at doses ranging from 25-75 mg/m²at 21 day intervals for adriamycin, to 35-50 mg/m² for etoposideintravenously or double the intravenous dose orally.

Agents that disrupt the synthesis and fidelity of nucleic acidprecursors and subunits also lead to DNA damage. As such a number ofnucleic acid precursors have been developed. Particularly useful areagents that have undergone extensive testing and are readily available.As such, agents such as 5-fluorouracil (5-FU), are preferentially usedby neoplastic tissue, making this agent particularly useful fortargeting to neoplastic cells. Although quite toxic, 5-FU, is applicablein a wide range of carriers, including topical, however intravenousadministration with doses ranging from 3 to 15 mgkg/day being commonlyused.

Thus, specific chemotherapeutic agents contemplated to be of used,include, e.g., adriamycin, 5-fluorouracil (5FU), etoposide (VP-16),camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP) nitrosoureas(BCNU, CNU) and even hydrogen peroxide, MMS, mafosfamide, thiotepa, freeradical radiomimetics such as bleomycin, and antimetabolites such asAra-C. The invention also encompasses the use of a combination of one ormore of these agents with radiation-based treatments, such as the use ofX-rays, with the further addition of APE down-regulation or inhibitionof activity. In certain embodiments, the use of topoisomerase IIinhibitors such as VP-16 and camptothecinin in combination withradiation and antisense APE expression are particularly preferred.

In addition to chemo- and radiotherapies, it also is contemplated thatcombination with gene therapies will be advantageous. For example, p53or p16 are powerfiul tumor suppressors that can be used effectively astherapeutics. Any other tumor-related gene conceivably can be targetedin this manner, for example, p21, Rb, APC, DCC, NF-1, NF-2, BCRA2, p16,FHIT, WT-1, MEN-I, MEN-II, BRCA1, VHL, FCC, MCC, ras, myc, neu, raf;erb, src, fins, jun, trk, fos, ret, gsp, hst, bcl and abl, using eithera sense or antisense approach.

The skilled artisan is directed to "Remington's Pharmaceutical Sciences"15th Edition, chapter 33, in particular pages 624-652. In this regard,some classes of agents include alkylating agents such as nitrogenmustards, ethylenimines and methylmelamines, alkyl sulfonates,nitrosoureas, triazenes, cyclophosphoramide, and the like;antimetabolites such as folic acid analogues, pyrimidine analogues, inparticular fluorouracil and cytosine arabinoside, and purine analoguesand the like; natural products such as vinca alkaloids,epipodophyllotoxins, antibiotics, enzymes and biological responsemodifiers; and miscellaneous products such as platinum coordinationcomplexes, anthracenedione, substituted urea such as hydroxyurea, methylhydrazine derivatives, adrenocorticoid suppressants and the like. Somevariation in dosage will necessarily occur depending on the condition ofthe subject being treated. The person responsible for administrationwill, in any event, determine the appropriate dose for the individualsubject. Moreover, for human administration, preparations should meetsterility, pyrogenicity, general safety and purity standards as requiredby FDA Office of Biologics standards.

The inventor proposes that the regional delivery of APE-inhibitoryagents to patients will be a very efficient method for delivering atherapeutically effective gene to counteract the clinical disease.Similarly, the chemo- or radiotherapy may be directed to a particular,affected region of the subjects body. Alternatively, systemic deliveryof the APE-regulating compound and/or the secondary agent may beappropriate in certain circumstances.

These compositions all would be provided in a combined amount effectiveto induce apoptosis in a cell. This process may involve contacting thecells with the APE-related agent(s) and other factor(s) at the sametime. This may be achieved by contacting the cell with a singlecomposition or pharmacological formulation that includes both, or bycontacting the cell with two distinct compositions or formulations, atthe same time, wherein one composition includes the expression constructand the other includes the agent.

Alternatively, the anti-APE therapy treatment may precede or follow theother agent treatment by intervals ranging from minutes to weeks. Inembodiments where the other agent and anti-APE therapy are appliedseparately to the cell, one would generally ensure that a significantperiod of time did not expire between the time of each delivery, suchthat the agents would still be able to exert an advantageously combinedeffect on the cell. In such instances, it is contemplated that one wouldcontact the cell with both modalities within about 12-24 hours of eachother and, more preferably, within about 6-12 hours of each other, witha delay time of only about 12 hours being most preferred. In somesituations, it may be desirable to extend the time period for treatmentsignificantly, however, where several days (2, 3, 4, 5, 6 or 7) toseveral weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respectiveadministrations.

It also is conceivable that more than one administration of either theanti-APE or the other agent will be desired. Various combinations may beemployed, where anti-APE is "A" and the other agent is "B", asexemplified below:

    __________________________________________________________________________    A/B/A         B/A/B              B/B/A                   A/A/B                        B/A/A                             A/B/B                                  B/B/B/A                                       B/B/A/B    A/A/B/B         A/B/A/B              A/B/B/A                   B/B/A/A                        B/A/B/A                             B/A/A/B                                  B/B/B/A    A/A/A/B         B/A/A/A              A/B/A/A                   A/A/B/A                        A/B/B/B                             B/A/B/B                                  B/B/A/B    __________________________________________________________________________

Other combinations are contemplated. Again, to achieve induction ofprogrammed cell death, both agents are delivered to a cell in a combinedamount effective to induce apoptosis.

C. Reestablishing Normal APE Levels in APE-Deficient Cells

In other embodiments, the present invention provides a method fortreating cells to prevent or render the cells less susceptible toapoptosis, which involves the step of increasing the amount of APEactivity or protein in the cells. This may be particularly useful inreestablishing normal APE levels in cells that have normal or abnormalcell senescence and/or death is occurring. For example, the reduction ofcertain T-cell populations in immunodeficiency diseases are thought toinvolve apoptotic fuinctions and many of these apoptotic events may beabrogated by the present invention. Similarly, IFNβ overproduction isthought to render circulating memory T cells competent to apoptosis byupregulating the cascade of metabolic events leading to programmed celldeath.

The present invention may be able to circumvent such premature celldeath that is associated with various immunodeficiencies by supplying afinctional APE. This may be achieved by, for instance, by selectiveactivation or overexpression of an APE gene in the cell which expressesAPE. In the alternative, it may be possible to use genetic therapy withAPE-expressing vectors, described above, to increase levels of APE issuch cells.

D. Monitoring Cancer Therapies

As demonstrated in the Examples (below), the level of APE drops in cellsthat are preparing to undergo apoptosis. One scenario in which cells areundergoing programmed cell death is as part of a cancer therapy. Thus,the present invention contemplates use of the above-described diagnosticmethodologies to measure the efficacy of standard cancer therapies byvirtue of a related drop in APE level.

The cancer to be treated may be virtually any type of cancer includingcancers of the brain, lung, prostate, cervix, liver, spleen, kidney,lymph node, small intestine, pancreas, blood cells, colon, stomach,breast, endometrium, testicle, ovary, skin, head and neck, esophagus,bone marrow and blood. While some malignancies may exhibit an increasein APE levels, it is not necessary that the cancer exhibit increased APElevels in order for the monitoring to have value.

In one embodiment, the clinician would take a sample of the tumor tissueprior to exposure of the patient to the therapy. A determination of APElevels would provide a base-line for measurement of that tissue'sexisting APE expression. It also is contemplated that "normal" levelsfor the corresponding normal tissue would be known by virtue ofmeasuring that tissue type from a statistically significant group ofindividuals. Thus, both absolute and relative base-lines would beavailable.

During a therapeutic regimen, additional samples would be taken todetermine the effect of the therapy on the APE levels in the tumor and,hence, the likelihood that apoptosis was induced. Based on the type andlocation of the tumor, the type and duration of treatment, and thehealth state of the patient, the sampling will vary.

E. Immunotherapies

Immunotherapeutics, generally, rely on the use of immune effector cellsand molecules to target and destroy abnormal cells. The immune effectormay be, for example, an antibody specific for some marker on the surfaceof a cancer cell. The antibody alone may serve as an effector of therapyor it may recruit other cells to actually effect cell killing. Theantibody also may be conjugated to a drug or toxin (chemotherapeutic,radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) andserve merely as a targeting agent. Alternatively, the effector may be alymphocyte carrying a surface molecule that interacts, either directlyor indirectly, with a tumor cell target. Various effector cells includecytotoxic T cells and NK cells.

According to the present invention, it is unlikely that APE could serveas a target for an immune effector given that it is unlikely to beexpressed on the surface of the cell. However, it is possible that APEmay be targeted by immunotherapy using antibodies modified to be takenup by target cells or by a single-chain antibody expression construct.

F. Protein Therapy

Another therapy approach is the provision, to a subject, of an APEpolypeptide, active or inactive APE fragment, APE synthetic peptide, APEmimetic or other analogs thereof. The protein may be produced byrecombinant expression means or, if small enough, generated by anautomated peptide synthesizer. Formulations would be selected based onthe route of administration and purpose including, but not limited to,liposomal formulations and classic pharmaceutical preparations.Targeting moieties also may be included in the preparations to aid inthe targeting of particular cells and in the uptake of the protein bythe target cells.

G. Formulations and Routes for Administration to Patients

Where clinical applications are contemplated, it will be necessary toprepare pharmaceutical compositions--expression vectors, virus stocks,proteins, antibodies and drugs--in a form appropriate for the intendedapplication. Generally, this will entail preparing compositions that areessentially free of pyrogens, as well as other impurities that could beharmful to humans or animals.

The agents can be administered orally, intravenously, intramuscularly,intrapleurally or intraperitoneally at doses based on the body weightand degree of disease progression of the patient, and may be given inone, two or even four daily administrations.

One will generally desire to employ appropriate salts and buffers torender agents stable and allow for uptake by target cells. Aqueouscompositions of the present invention comprise an effective amount ofthe agent, dissolved or dispersed in a pharmaceutically acceptablecarrier or aqueous medium. Such compositions also are referred to asinocula. The phrase "pharmaceutically or pharmacologically acceptable"refer to molecular entities and compositions that do not produceadverse, allergic, or other untoward reactions when administered to ananimal or a human. As used herein, "pharmaceutically acceptable carrier"includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. The use of such media and agents for pharmaceutically activesubstances is well know in the art. Except insofar as any conventionalmedia or agent is incompatible with the vectors or cells of the presentinvention, its use in therapeutic compositions is contemplated.Supplementary active ingredients also can be incorporated into thecompositions.

The active compositions of the present invention may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention will be via any common route so longas the target tissue is available via that route. This includes oral,nasal, buccal, rectal, vaginal or topical. Alternatively, administrationmay be by orthotopic, intradermal, subcutaneous, intramuscular,intraperitoneal or intravenous injection. Such compositions wouldnormally be administered as pharmaceutically acceptable compositions,described supra.

The active compounds may also be administered parenterally orintraperitoneally. Solutions of the active compounds as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial an antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freezedryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

As used herein, "pharmaceutically acceptable carrier" includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

For oral administration the polypeptides of the present invention may beincorporated with excipients and used in the form of non-ingestiblemouthwashes and dentifrices. A mouthwash may be prepared incorporatingthe active ingredient in the required amount in an appropriate solvent,such as a sodium borate solution (Dobell's Solution). Alternatively, theactive ingredient may be incorporated into an antiseptic wash containingsodium borate, glycerin and potassium bicarbonate. The active ingredientmay also be dispersed in dentifrices, including gels, pastes, powdersand slurries. The active ingredient may be added in a therapeuticallyeffective amount to a paste dentifrice that may include water, binders,abrasives, flavoring agents, foaming agents, and humectants.

The compositions of the present invention may be formulated in a neutralor salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms such as injectable solutions, drug release capsules and thelike. For parenteral administration in an aqueous solution, for example,the solution should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, "Remington's PharmaceuticalSciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

VIII. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well within thepractice of the invention, and thus can be considered to constitutepreferred modes for of practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

Example 1 Materials And Methods

Antibody production: For overexpression of glutathioneS-transferase:human APE fusion protein (GST-APE), bacterial cultures (10ml) containing the human GST-APE fusion constructs were grown overnightat 37° C. in LB plus 100 μg/μl ampicillin, generally as describedpreviously for other DNA repair genes (Wilson et al., 1996; 1994).Overnight cultures were diluted 1 to 10 in fresh, pre-warmed (37° C.) LBmedium supplemented with ampicillin and grown for 1 hour at 37° C. withshaking. Expression of the GST-APE fusion protein was induced by addingisopropyl-β-D-thio-galactoside (IPTG) to a final concentration of 0.1 mMand growing the cells for an additional 3.5 hours at 37° C. Cells wereharvested by centrifugation at 1000×g for 10 minutes and washed oncewith PBS, pH 7.4. Packed cells were resuspended in 3 ml of PBS and lysedby mild sonication (two 20-second bursts) on ice. The human APE fusionprotein was totally soluble. Cellular debris was pelleted bycentrifugation at 8500×g for 10 minutes at 4° C. and the supernatantcollected containing the soluble human APE fusion protein. The solublefraction had Triton X-100 added to a final concentration of 0.1% andloaded onto a glutathione Sepharose 4B column, pre-equilibrated withPBS.

Binding of the GST-APE protein was carried out for 3 hours on a nutatorat 4° C. The column was subsequently washed with 20 column volumes ofPBS containing 0.1% Triton X-100. Following the wash, the fusion proteinwas eluted with 50 mM Tris, pH 7.5, containing 10 mM glutathione,fractions collected and analyzed by SDS-polyacrylamide gelelectrophoresis. This column purified protein was cleaved from theglutathione-S-transferase portion using Factor Xa. The clipped, gelpurified proteins were used as antigen for polyclonal antibodyproduction in rabbits and for preabsorbing the antibodies to confinspecificity in immunohistochemistry or Western blot experiments.

SDS-Polyacrylamide Gel Electrophoresis, Antibody Production,Electroelution of Protein from Gels and Western Blot Analysis: Proteinsamples were fractionated on a 12% SDS-polyacrylamide gel. For isolationof gel-purified antigen, column purified GST-APE fusion protein wasfirst cleaved with Factor Xa and the products resolved on a 12%SDS-polyacrylamide gel. The clipped APE protein was excised from the geland this gel strip placed into dialysis tubing with 1 ml of 1× SDSelectrophoresis buffer (0.125 M Tris-HCl; 0.96 M glycine; 0.5% SDS). Thetubing was sealed and the antigen eluted in 1× SDS electrophoresisbuffer at 150 V for 20 min. This protein was used for both antigenproduction and immunoabsorption experiments.

In particular, the anti-human apurinic/apyrimidinic endonucleaseantibody, anti-APE, was obtained by injection of rabbits with about 15mg of each (native and denatured) purified GST-APE fusion proteinpreparation (Harlow and Lane, 1988). Animals were initially injectedwith the antigen in an emulsion containing equal volume of Hunter'sTiterMax. Preimmune serum was collected at the time of the firstinjection using the ear bleed method. Red blood cells were removed fromthe serum by incubation at 4° C. overnight and centrifugation at 3000×gfor 10 minutes. Serum (the supernatant) was stored in aliquots at -20°C. Rabbits were anesthetized prior to injections and bleeds withketamine (35 mg/kg) and rompun (5 mg/kg). Animals were boosted every 3to 5 weeks with an emulsion containing an equal volume of antigen andincomplete Freund's adjuvant. At this time, anti-APE serum was harvested(5 ml of blood/1b).

For affinity purification of anti-APE, approximately 5 mg of purifiedGST-APE fusion was electrophoresed on a 12% SDS-polyacrylamide gel andblotted onto a nitrocellulose membrane. The region of nitrocellulosewhich corresponded to the location of the fusion protein was cut into astrip and used to affinity purify the antibody (Harlow and Lane, 1988;Maniatis et al., 1989). The filter strip was blocked with Blotto for 1hour at room temperature and incubated with 3 ml of anti-APE serum on arotator at 4° C. overnight. The filter was washed 3 times with 2× TBSTfor 10 minutes each. To elute the antibody, the strip was covered in aminimal volume of Elution Buffer (Pierce), placed in a damp chamber andincubated for 20 minutes on the rotator at room temperature. The ElutionBuffer was passed over the strip several times and transferred to alabeled tube for storage at 4° C. The sample was subsequently dialyzedagainst PBS, pH 7.4 for 18 hours and termed affinity purified anti-APE.

Western blot analysis was performed as has been previously described(Wilson et al., 1994; 1995, FIG. 16). Whole cell extracts wereelectrophoresed on a 12% SDS-PAGE and electroblotted onto 0.2 micronnitrocellulose. The filter was incubated in blocking buffer whichcontains 5% non-fat milk for 1 hour. Next, the antibody (affinitypurified APE) was diluted to the appropriate concentration (1:100) andincubated with the filter overnight at 4° C. The filter was washed in 1×TBST and incubated in TBST containing 5% milk and cross-reactingproteins were detected using the Chemiluminescence Western Blotting Kitfrom Boehringer-Mannheim (Indianapolis, Ind.) as directed. Western blotanalysis of HeLa cell (human) or NIH3T3 (mouse) cells using antibodyproduced in rabbits or chickens are shown in FIG. 16. The antibodieswere affinity purified and only a single cross-reacting protein isvisible in the human or mouse cells.

The inventor has compared the efficacy of the APE polyclonal made hereinto a commercially available polyclonal and five monoclonal antibodiesthat have not yet been published. Using Western blot analysis (FIG. 20),the inventor's APE antibody gives much more specific cross-reactivity toAPE protein in HeLa cells compared to the commercially availableantibody, i.e., only one cross-reacting band observed. Of the fivemonoclonal antibodies, only A1 gives a clean signal upon Western blotanalysis. Furthermore, the given monoclonal antibodies do not give adetectable signal when used in immunohistochemistry assays and thecommercial APE polyclonal does not give a clean signal in IHC. Theinventor would be reluctant to use the commercial antibody due to itsobvious cross-reactivity to multiple bands as evidenced upon Westernblot analysis (FIG. 20).

Example 2 Biopsy Samples

Paraffin-embedded biopsies from over thirty cases of squamous cellcarcinoma in situ and squamous cell carcinoma of the cervix wereobtained from the archives of University Hospital and Wishard MemorialHospital, Indianapolis, Ind. This study was approved by the IndianaUniversity Institutional Review Board. Six micron sections were stainedwith rabbit anti-APE as described above and in (Duguid et al., 1995).Cervical tissue was obtained which had been previously evaluated forcervical cancer or normal controls. The tissues were classified asnormal, dysplastic (mild, moderate, severe), cervical intraepithelialneoplasia (CIN) or SCC grade 2 (SCC-G2) or grade 3 (SCC-G3). All sampleswere analyzed using a blind coding system, such that the antibodystaining was performed on numbered slides and the sample diagnosis wasnot known at the time of processing. Although only representative dataare presented in FIGS. 3 and 4, the results have been confirmed withover thirty other cervical cancer tissue samples. Immunohistochemicalanalysis was performed as previously described (Wilson et al., 1995;Duguid et al., 1995). Briefly, primary anti-APE antibody (rabbitanti-human APE polyclonal) was incubated with the section overnight at4° C. at a 1:50 dilution in 10% goat serum in PBS. The following day,the sections were washed three times for 5 minutes in PBS followed byincubation with the secondary antibody (biotinylated goat anti-rabbitIgG, Vector Labs, Burlingame, CA) at 15 μg/ml in 10% goat serum for 1hour. Following two 5 minute PBS washes, the sections were incubatedwith aviden and biotinylated horseradish peroxidase complex (ABC elitekit, Vector Labs) for 45 minutes. The sections were then incubated withthe chromogen diaminobenzidine (Vector Labs). After development ofsignal, the sections were washed briefly in dH₂ 0 and dehydrated througha graded alcohol series to xylene, coverslipped, analyzed andphotographed. To control for antibody specificity, preimmune IgG wasused as the primary antibody in place of the anti-APE antibody at aconcentration of 50 μg/ml. for histological staining, the adjacentsections were stained with hematoxylin and esoin and coverslipped(Duguid et al., 1995; Wilson et al., 1995).

The subcellular distribution and level of APE was examined in thirtybiopsies of various stages of cervical cancer, including mild to severedysplasia, carcinoma in situ and squamous cell carcinoma usingimmunohistochemistry, and it was found that in all cases of premalignantand malignant tissue, increased levels of APE were detected compared tonormal control tissues (FIG. 3).

The specificity of the APE antibody on cervical cell line extracts wasconfirmed using Western blot analysis of three HIV+ cervical cell lines(CaSki, HeLa and SiHa) (FIG. 1). A single cross-reacting band at Mr37,000 was observed as expected. The specificity of the APE antibody wasalso confirmed as it detected the GST-APE fusion protein from E. coliextract of the recombinant overproducing strain. The applicants havefound only a single cross-reacting band found in the C33a cell linewhich is HPV-, but has mutated p53 and retinoblastoma genes. HeLacontains HPV-18, SiHa contains HIV-16 (102 copies/cell) and CaSki alsocontains HPV-16, but with approximately 600 copies per cell. Since afull 85% of patients with cervical cancers are PHV positive, these celllines were chosen as representative models of this phenomena (Park etal., 1995; Schiffman et al., 1995). The HPV- C33a cell line was aninitial attempt to determine if there was any relationship to APEexpression levels and HPV. No relationship has been observed in work todate.

The subcellular location of APE in cervical cancer cell lines wasdetermined and compared with the actual human cervical tissues. The APEantibody predominantly demonstrated cross-reactivity in the nuclei ofthe four cell lines (FIG. 2). There did not appear to be much detectionof protein in the cytoplasm of the cells, nor was there any immediatelyobservable differences between the three HPV+ (HeLa, CaSki and SiHa) andHPV- (C33a) cell lines. A rather distinctive punctate staining of thenucleus was observed. The nuclear localization was confirmed using humannormal and cervical tissues and is presented below.

The subcellular distribution of APE was examined in a variety ofcervical tissues, spanning the range of cervical cancer progression,including normal controls, mild to severe dysplasia, CIN, and SCC-G2 andSCC-G3. Representative immunohistochemical stainings of some of thesenormal and cervical cancer conditions are shown in. FIGS. 3 and 4. Thelevel of APE protein is consistently increased compared to normalcervical tissue, beginning with the mild dysplasia and dramaticallyelevated in the SCC-G2 and SCC-G3 (FIG. 3). Althoughimmunohistochemistry is not as quantitative as other types of analysissuch as Western blotting of proteins or in situ hybridization for mRNAlevels, it can be used for comparative analysis.

The increase in the APE protein appears to be mainly nuclear; however,upon close examination of a large number of samples, there also appearsto be an increase in the level of APE that is both nuclear andcytoplasmic in some cells (FIG. 4E). This may just be an overallincrease in APE protein expression or it may be the result of a decreasein trafficking into the nucleus.

Example 3 Cervical Cancer Evaluation for APE Staining

Table 4 depicts cervical cancer evaluation data. Two aspects of thestaining were scored. First, an estimation of the proportion of cellsstaining and the intensity of the nuclear staining from 0 to 3. Anycells staining less than 10% of the cells positive was considerednegative (0).

First, the immunostain was looked at, various different patterns ofstaining were selected, up to three, and then correlated these areaswith the H&E stain. Interpretation of these specific areas withoutknowing what the case actually signed out was determined. On each slide,there may be up to three diagnosis (Dx), followed by the staininterpretation for that specific area.

Regions of the epithelium staining were basal layer (BL), Basilar 1/2(B1/2) and Superficial 1/2 (S1/2). these regions do not apply with theinvasive squamous cell lesions.

There were only a few discrepancies in the interpretation versus thesign out, but no significant difference. Further, staining followedpathology and APE staining appears to be more predictive and a marker ofearlier stages in dysplasia and CINI, etc. and not as much on the veryfull blown SCCC. Therefore it may be a much better marker for earlierand premalignant stages of cervical cancer.

                  TABLE 4    ______________________________________    Pathology Diagnosis               Number of Cases                           APE Ab Diagnosis*    ______________________________________    CIN 1      13          12-CIN 1; 1-CIN 3    CIN 2      10          8-CIN 2; 2-CIN 1    CIN 3      7           4-CIN 3; 3-CIN 2    SCC G1     3           3-CIN 3    SCC G2     7           5-SCC G2; 2-CIN 3    SCC G3     5           5-SCC G3    Normal     10          10 Normal, low level of staining    Total      55    ______________________________________

The actual staging differed in some cases, but this assumes thepathology grading was accurate. No cases went undetected.

Example 4 APE in Prostate Cancer Tissue

Cancerous prostate tissue was stained in accordance with the protocolsdescribed above for the cervical tissue. The results, discussed aboveand presented in FIG. 5A-C as well as FIG. 18A-C and FIG. 19A and FIG.19B demonstrate that elevated levels of APE are also an indicator forcancerous prostate tissue.

APE has also been found to be elevated in cancerous prostate tissue, andthus can similarly be used as a marker to identify cancerous states inthe prostate. In particular, FIG. 5 shows immunohistochemical stainingof prostate tissue, in which FIG. 5A. is hematoxylin and eosin stainingof prostate tissue. FIG. 5B shows cancerous prostate cells (closedarrows) bordering normal tissue (open arrows). The level of APE ishighly elevated in the prostate cancer. FIG. 5C. shows invasion ofprostate nerve tissue by the cancer (filled arrows).

FIG. 18A, FIG. 18B and FIG. 18C also show prostate tissue with APE. FIG.18A is a lower magnification (10×) of a section of prostate tissueshowing normal (arrowheads) and cancer cells (arrows with tails). FIG.18B is higher magnification (40%) of cancer cells from FIG. 18A showinga strong nuclear staining in a punctuate manner. FIG. 18C. low powermagnification of prostate tissue with cancerous region to lower left(below arrows) and normal cells in upper right region. Distinctivehigher levels of APE staining in the cancerous region can be seen.

In a further study, tissue samples were collected from the surgicalpathology files of University Hospital at Indiana University School ofMedicine. All tissue samples were fixed in 10% buffered formalin,embedded in paraffin and sectioned at 6 μm thickness. The prostatesamples that contained malignant neoplasm were selected from cases ofpreviously diagnosed tumors treated with prostectomy. The cases withbenign lesions were obtained from transurethral resection specimens.

A semi-quantitative assessment of the specimen staining characteristicwere evaluated as well as observation of the microscopic pattern. Thespecimens stained with antibody against APE protein were examined firstwithout knowledge of diagnosis or microscopic features of the histologicslides. Both nuclear and cytoplasmic staining were noted. Anyappreciable brown staining was considered positive and graded as 1+ ifbarely detectable; 2+ if easily seen fine granules were presentdiffusely throughout the nucleus or cytoplasm, and 3+ when dark coursegranules were observed. Also, the percent of cells exhibiting positivestaining were estimated. Less than 10% of the cells show presence ofstain were considered negative; 10%-30% of the cells positive weregraded as 1+, 30%-60% as 2+ and >60% were graded as 3+. The nuclear andcytoplasmic staining were recorded separately. The H&E slides were thenreviewed to determine diagnosis and map the location of the varioushistologic patterns such as carcinoma, glandular hyperplasia and normalto correlate with the staining patterns observed in theimmunohistochemical preparations.

There was a distinct contrast in the staining characteristics betweenthe areas containing malignant neoplasm and those containing benignglands (Table 5). The vast majority of the malignant tumors showedstrong nuclear and cytoplasmic staining, whereas the benign areas withinthe slides containing adenocarcinoma as well as the glands of the caseswith only benign diagnosis, showed minimal to negative staining. Five ofthe nine adenocarcinomas, exhibited 3+ nuclear staining in greater than60% of the cells with similar cytoplasmic staining. One of the cases ofadenocarcinoma had 2+ nuclear positivity and 2+ cytoplasmic staining.Only one exhibited 1+ nuclear staining, but it had 3+ cytoplasmicstaining. Two of the malignant tumors showed negative nuclear staining,but one of these had 3+ cytoplasmic staining. The other exhibited totalnegative staining, raising the possibility of technical artifact.Conversely, the benign areas within the slides containing malignancywere generally negative with only rare foci showing 1+ to 2+ staining ofthe nuclei and cytoplasm. One of the slides consisting of glandularhyperplasia exhibited 2+ nuclear staining of 30% to 60% of the cells butexpressed no cytoplasmic staining. One case of the adenocarcinoma (case1120) showed an interesting strong punctuate supranuclear cytoplasmicstaining pattern with relatively weaker nuclear staining. Also, few fociof glands with an atrophic appearance expressed strong nuclear staining.Case 835 had transitional cell carcinoma which was thought to be a goodcomparison with the other cases. It showed strong nuclear staining andthe transitional tumor cells also extended into the prostate and wasclearly visible (FIG. 19A and FIG. 19B).

                  TABLE 5    ______________________________________                                        Cancer                                              Normal    No.   Case    Year    Diagnosis                                  Tissue                                        Cells cells    ______________________________________    1     144     1994    G2 (2 + 3)                                  Prostate                                        3+    0    2     633     1994    G2 (4 + 5)                                  Prostate                                        2+    0    3     6488    1995    G2 (2 + 3)                                  Prostate                                        3+    1+    4     6496    1995    G2 (3 + 3)                                  Prostate                                        N/A   N/A    5     315     1994    G2 (3 + 4)                                  Prostate                                        3+    0    6     597     1995    hyperplasia                                  Prostate                                        N/A   N/A    7     1120    1995    G3      Prostate                                        2+    0    8     835     1995    TCC/Nod.                                  Prostate                                        3+                          Hyperplasia    9     1157    1995    G2      Prostate                                        3+    0    10    1931    1995    G2      Prostate                                        3+    0    11    1687    1995    G2      Prostate                                        2+    0    12    835     1995    TCC     Prostate                                        3+    0    ______________________________________

Example 5 APE Expression in Differentiating Myeloid Leukemia Cells

To investigate possible changes in expression of the DNA repair enzymeAPE in differentiating myeloid cells, the inventor utilized Northern andWestern blot analysis with the HL-60 myeloid leukemia cell line, whichcan be induced to terminally differentiate into mature granulocytes ormonocyte/macrophage (Collins, 1987). Western blot analysis revealed aprogressive decrease in APE protein expression over 6 days in HL-60cells induced to differentiate along the granulocytic pathway with 10⁻⁵M RA compared to ethanol treated controls (FIG. 6A). A more rapiddecrease in APE expression was observed with granulocytic inductionusing DMSO (FIG. 6A) with nearly undetectable APE after 6 days. Theseobservations were confirmed by Northern blot analysis (FIG. 6A) where asimilar progressive decrease in APE transcript expression was observedwith induction of granulocytic differentiation. Again DMSO induces amore rapid decrease to a lower level compared to RA. To determinewhether the decrease in APE expression was confined to granulocyticdifferentiation, HL-60 cells were induced down the monocyte/macrophagepathway with PMA. Expression of the APE protein decreased to a very lowlevel by Western blot (FIG. 6B) after two days exposure to PMA.Decreased expression of the APE transcript on Northern blot was alsoevident after exposure to PMA (FIG. 6B). Thus, induction of granulocyticand monocytic differentiation in HL-60 cells results in decreasedexpression of APE at the RNA and protein level.

Example 6 Overexpression of the bcl-2 oncogene in HL-60 cells inhibitsapoptosis does not affect retinoic acid induced differentiation

To determine whether cell differentiation and apoptosis were closelylinked processes that could not be separated, the inventor overexpressed the proto-oncogene bcl-2 in HL-60 cells to block apoptosis.the 1.9 kB cDNA of bcl-2 was subdloned into the retroviral vector LXSNto produce Lbcl2SN. The ecotropic packaging cell line PE501 wastransfected and used to transduce the amphotropic packaging cell linePA317. High titer clones of PA317+Lbcl2SN were selected for transductionof HL-60 cells to produce HL-60+Lbcl2SN. Several clones were screened byNorthern blot analysis and one clone with high bcl-2 expression waschosen for study. Treatment of the HL-60 +Lbcl2SN cells with RA resultedin well differentiated mature granulocytes (metamyelocytes, myelocytes,bands and neutrophils) expressing CD11b (Table 6). These cells wereassayed for programmed cell death using the TUNNEL reaction to scoreindividual cells and the diphenylamine assay to determine the totalamount of DNA undergoing fragmentation. The transduced HL-60+Lbcl2SNcells treated with RA had a much lower level of apoptosis (FIG. 10A andFIG. 10B) and enhanced viability with survival of mature granulocytes upto 40 days compared to the parental HL-60 cells treated with RA (FIG.11A and FIG. 11B). Thus myeloid cell differentiation and apoptosis areindependent processes that can be separated.

                  TABLE 6    ______________________________________    RA-Induced Differentiation of HL-60 and HL-60-bcl-2 Cells                HL-60      HL-60-bcl-2                (-) RA                      (+) RA   (-) RA   (+) RA    ______________________________________    Mature myeloid cells (%).sup.1                  <5      86 = 6   7 = 3  88 = 5    CD11b (+).sup.2                  69.2    105.4    59.1   107.9    NBT (+).sup.3 <5      >90      5      >90    ______________________________________     Cells were induced with RA (1 mmol/L) for 7 days and evaluated by     morphology. CD11b surface antigen expression. and NBT reduction.     .sup.1 Myelocytes, metamyelocytes, banded and segmented granulocytes on     WrightGiemsa-stained preparations of cell suspensions. Numbers represent     the average percentage: The observed range in triplicate experiments.     .sup.2 Results are expressed as the relative mean fluorescence on an     arbitrary log scale from 0 to 200 where an increment of 18.5 U represents     a doubling of fluorescent intensity. Data on 5,000 cells were analyzed fo     each sample.     .sup.3 Percentage of cells containing blueblack formazan deposits after 1     hour of TPA stimulation.

Example 7 APE Expression and bcl-2 Expression

To ascertain whether APE expression was associated with differentiationor programmed cell death, the inventor blocked apoptosis byover-expression of the proto-oncogene bcl-2 using HL-60 cells transducedwith the retroviral construct Lbcl2SN (Park et al., 1994). Western blotanalysis of the HL-60-bcl-2 cells induced down the granulocytic pathwaywith RA or DMSO revealed continuous expression of APE compared to therapid decrease in expression observed with similar treatment of theparental HL-60 cells (FIG. 6A and FIG. 7A). Expression of APE mRNA inthe HL-60-bcl-2 cells after exposure to RA or DMSO is similarlymaintained paralleling the protein expression (FIG. 7A). The apparentdecrease in APE transcript expression after 6 days exposure to RA orDMSO results from loading differences as evidenced by the GAPDH Northerncontrol blot (FIG. 7). Monocyte/macrophage differentiation induction ofHL-60-bcl-2 cells with PMA also revealed continuous expression of APEboth at the protein and RNA level (FIG. 7B). Thus constitutiveexpression of bcl-2 inhibits the decrease in APE expression as well asprogrammed cell death normally observed with granulocytic ormonocytic/macrophage induced differentiation of HL-60 cells.

Example 8 APE Expression And Programmed Cell Death

The correlation between the decreased expression of APE in induced HL-60cells and programmed cell death was examined by quantitating the numberof cells undergoing apoptosis in induced HL-60 and HL-60-bcl-2 cellsusing the in situ TUNNEL assay. There is a baseline low incidence (1-2%)of apoptosis in HL-60 cells that does not change following ethanol (FIG.8) and probably reflects the few HL-60 cells that can be observed tospontaneously differentiate in untreated growing cultures.Over-expression of bcl-2 depresses the baseline apoptosis to less than1% (FIG. 8). Granulocytic differentiation induction (RA, DMSO), as wellas monocyte/macrophage differentiation induction (PMA) resulted in astatistically significant inhibition of apoptosis by day 6 in theHL-60-bcl-2 cells compared to the wild type HL-60 cells (FIG. 8). Thehigh level expression of bcl-2 in HL-60-bcl-2 cells appears to beresponsible for not only the block in apoptosis (Park et al., 1994), butalso the failure to downregulate expression of APE with induction ofdifferentiation. Hence, the bcl-2 transduced HL-60 cells differentiate(RA, DMSO, or PMA) morphologically but do not undergo apoptosis and donot exhibit decreased expression in APE, thus establishing the inverserelationship between programmed cell death and APE expression. Thisleads to the conclusion that the decrease in expression of APE observedon Northern and Western blot (FIG. 6) in induced HL-60 cells isassociated with programmed cell death.

To determine if the cells undergoing apoptosis were also the cellsdownregulating APE, double labeling experiments were performed on a50:50 mix of untreated and RA treated (6 days, 10⁻⁵ M) HL-60 cells (FIG.9A). Cytocentrifuge preparations were stained for 1) fragmented DNAusing fluorescein-dUTP in the TUNEL assay (FIG. 9C) and 2) APE usingpolyclonal rabbit anti-APE with a rhodamine labeled goat anti-rabbitsecondary antibody (FIG. 9B). Cells undergoing apoptosis fluoresce`green` and cells expressing APE fluoresce `red`. Examination of thecells revealed that cells staining positive with the TUNEL assay, thusundergoing apoptosis, had little or no APE as evidenced by the absenceof rhodamine fluorescence. Conversely, TUNEL negative cells, notundergoing apoptosis, stained strongly positive for APE. Thus, HL-60cells undergoing apoptosis appear to lose expression of APE.

The inventor further characterized the temporal decline in APEexpression to see if it was an early event in programmed cell death orif the decline coincided with fragmentation of DNA (TUNEL assay); theinventor assayed APE expression and DNA fragmentation on cytospunpreparations of cells after exposure to DMSO. The expression of APEstarted to fall with fewer rhodamine positive cells after 2 days of DMSOwhile statistically significant evidence of fragmented DNA was notapparent until after 4-6 days (Table 7, FIG. 8). A similar pattern wasobserved, but not as rapid a loss of APE expression, after exposure toRA or PMA. Thus the decrease in APE expression associated withprogrammed cell death appears to be an early event occurring before thefinal pathologic consequences of DNA fragmentation in apoptosis.

                  TABLE 7    ______________________________________    Temporal relationship of AP endonuclease expression to apoptosis               % rhodamine    Cells      positive (APE)                           % fluorescein positive (TUNEL)    ______________________________________    HL-60 control               77%         1.7%    HL-60 + DMSO               34%         2.7%    2 days    HL-60 + DMSO                0%         2.3%    4 days    HL-60 + DMSO               ND           7%    6 days    ______________________________________

Therefore, the present study, associates apoptosis with downregulationof APE. The identification of a functional relationship and which APEdomains might be involved in programmed cell death awaits furtherexperiments will be elucidated with retroviral transduction of variousAPE constructs into cells as described in Example 6 and above.

Example 9 APE as a Marker of Apoptosis in Human cells DifferentiatingCD34.sup.± peripheral blood stem cells downregulate APE

The expression of APE in peripheral blood stem cells was examined to seeif APE expression would mimic what was observed with HL-60 cells.Peripheral blood mononuclear cells were obtained after informed consentby apheresis of volunteer adult subjects following 5 days of G-CSFmobilization. CD34+ cells were isolated using a MACS column. Highly pure(88+/-6% mean +/-SD, n=7) CD34+ cells were obtained with high recovery(77+/-7%, n=7). CD34+ cells were subsequently grown in the presence ofhuman stem cell factor (SCF) and interleukin (IL)-7 for lymphoiddifferentiation or SCF, IL-6 and G-CSF for myeloid differentiation.Adequate numbers of cells were collected at days 0, 3, 6 and 10 to allowanalysis both by Northern blots of RNA (probed with the human APE cDNA)and Western blots of protein using affinity purified antibody to humanAPE. FLOW analysis and morphologic examination confirmed thedifferentiated phenotype of the cells after culture. APE mRNA andprotein demonstrated a dramatic decrease during myeloid differentiationwith easily detectable levels present at day 0 falling to undetectablelevels after 10 days of differentiation. mRNA and protein levelscorrelated (FIG. 12A and FIG. 12B). A less dramatic fall in expressionof APE was seen in lymphoid culture conditions. Apoptosis, measured byFLOW and confirmed by TUNEL assay staining of cytospin preparations,increased during differentiation. The level of apoptosis inverselycorrelated with expression of APE, ie., was significantly higher inmyeloid cells. These data support the hypothesis that down-regulation ofAPE is an early event in cells undergoing apoptosis and reducedexpression of APE may explain the increased sensitivity of moredifferentiated myeloid cell populations versus hematopoietic stem cellsto alkylating agent cytotoxicity.

Example 10 Retroviral APE Constructs

As discussed above, recombinant retrovirus vectors are a highlyefficient method to introduce exogenous genes into hematopoietic cellsas well as cell lines. Myeloid cell lines, such as used by the inventorare often difficult to transfect with standard expression plasmids usingnormal molecular biology techniques. For that reason, as well as thepotential clinical use of the APE gene for gene transfer studies intoprimary hematopoietic cells, the inventor have developed recombinantretrovirus vectors to infect and express the recombinant APE gene andthe various permutations of the APE gene.

The retrovirus backbone that chosen is based on the Murine Stem CellVirus backbone that has been previously described (Hawley et al., 1993).The encoded genes are expressed from a myeloproliferative sarcoma virusLTR. The vector includes long gag sequences to promote higher titervirus production in retrovirus packaging cells, but contains mutationswhich interfere with gag-related protein synthesis and expression ofenv-related proteins. The constructs containing the human APE cDNAs arecompleted and an example is schematically shown in FIG. 13 and FIG. 14.

The retroviral plasmid is transfected into the packaging lines GP+E86and GP+AM12 (Markowitz et al., 1988a; Markowitz et al., 1988b). Thehelper virus genome of the packaging line is separated onto twoplasmids. The packaging signal and 3' LTR have been removed.Construction of these retroviral lines make the generation ofrecombinant retrovirus unlikely since at least three recombinationalevents must occur prior to generation of wild type virus. The GP+E86producer line contains a env gene which encodes for a viral envelopeprotein allowing for the infection of a narrow group of animals,specifically rodents such as mouse and rat (ecotropic range). TheGP+AM12 has an envelope protein that allows for the infection of a broadrange of animals including rodents and primates.

Individual packaging cell clones are isolated by selecting transfectedcells in 1 mg G418 powder/ml (GIBCO/BRL, Gaithersburg, Md.). IndividualG418 resistant clones are expanded and assayed for retrovirus productionas previously described (Clapp et al., 1995). Briefly, serial dilutionsof retrovirus supernatant from individual clones were assayed for theability to transmit G418 resistance (1 mg/ml) to NIH3T3 cells.Supernatant is collected following clonal expansion of producer cells toconfluence on plates and the addition of 8 ml of fresh media for 14-16hours collection. Supernatant is filtered through a 0.45 mm filter andlimiting dilutions of viral supernatant were added to 100 mm² plates ofNIH3T3 cells grown to approximately 30-40% confluence and cultured with6 μg/ml of polybrene for 2 hours followed by the addition of 8 ml ofmedia. The cells were then allowed to grow to confluence, split ontothree 100 mm² plates and selected in 1 mg/ml of G418.

In order to determine if the APE gene product can regulate or alter theendogenous APE gene in HL60 cells, the various APE constructs are usedto transduce HL60 cells. Cells are infected using virus supernatantinfections as previously described (Clapp et al., 1995) and thenselected in 1 mg/ml of G418 to remove all untransduced cells. Clones arepicked for insertion (Southern blot) and expression levels (Northems andWesterns) of the APE gene). Various clones can be picked with high,intermediate and low levels of retroviral APE production.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defmed by the appended claims.

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The following references are indicative of the level of skill in the artand are hereby incorporated herein by reference as if each had beenindividually incorporated by reference where cited above and fully setforth.

The following references are indicative of the level of skill in the artand are hereby incorporated herein by reference as if each had beenindividually incorporated by reference where cited above and fully setforth.

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    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 2    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1279 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    - GAATTCGGGG GTTGCTCTTT TGCTCATAAG AGGGGCTTCG CTGGCAGTCT GA - #ACGGCAAG      60    - CCGGTAAAAA TATTGCTTCG GTGGGTGACG CGGTACAGCT GCCCAAGGGG TT - #CGTAACGG     120    - GAATGCCGAA GCGTGGGAAA AAGGGAGCGG TGGCGGAAGA CGGGGATGAG CT - #CAGGACAG     180    - AGCCAGAGGC CAAGAAGAGT AAGACGGCCG CAAAGAAAAA TGACAAAGAG GC - #AGCAGGAG     240    - AGGGCCCAGC CCTGTATGAG GACCCCCCAG ATCAGAAAAC CTCACCCAGT GC - #GAAACCTG     300    - CCACACTCAA GATCTGCTCT TGGAATGTGG ATGGGCTTCG AGCCTGGATT AA - #GAAGAAAG     360    - GATTAGATTG GGTAAAGGAA GAAGCCCCAG ATATACTGTG CCTTCAAGAG AC - #CAAATGTT     420    - CAGAGAACAA ACTACCAGCT GAACTTCAGG AGCTGCCTGG ACTCTCTCAT CA - #ATACTGGT     480    - CAGCTCCTTC GGACAAGGAA GGGTACAGTG GCGTGGGCCT GCTTTCCCGC CA - #GTGCCCAC     540    - TCAAAGTTTC TTACGGCATA GGCGATGAGG AGCATGATCA GGAAGGCCGG GT - #GATTGTGG     600    - CTGAATTTGA CTCGTTTGTG CTGGTAACAG CATATGTACC TAATGCAGGC CG - #AGGTCTGG     660    - TACGACTGGA GTACCGGCAG CGCTGGGATG AAGCCTTTCG CAAGTTCCTG AA - #GGGCCTGG     720    - CTTCCCGAAA GCCCCTTGTG CTGTGTGGAG ACCTCAATGT GGCACATGAA GA - #AATTGACC     780    - TTCGCAACCC CAAGGGGAAC AAAAAGAATG CTGGCTTCAC GCCACAAGAG CG - #CCAAGGCT     840    - TCGGGGAATT ACTGCAGGCT GTGCCACTGG CTGACAGCTT TAGGCACCTC TA - #CCCCAACA     900    - CACCCTATGC CTACACCTTT TGGACTTATA TGATGAATGC TCGATCCAAG AA - #TGTTGGTT     960    - GGCGCCTTGA TTACTTTTTG TTGTCCCACT CTCTGTTACC TGCATTGTGT GA - #CAGCAAGA    1020    - TCCGTTCCAA GGCCCTCGCG AGTGATCACT GTCCTATCAC CCTATACCTA GC - #ACTGTGAC    1080    - ACCACCCCTA AATCACTTTG AGCCTGGGAA ATAAGCCCCC TCAACTACCA TT - #CCTTCTTT    1140    - AAACACTCTT CAGAGAAATC TGCATTCTAT TTCTCATGTA TAAAACGAGG AA - #TCCTCCAA    1200    - CCAGGCTCCT GTGATAGAGT TCTTTTAAGC CCAAGATTTT TTATTTGAGG GT - #TTTTTGTT    1260    #                 127 - #9    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 318 amino              (B) TYPE: amino acid              (C) STRANDEDNESS:              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    -      Met Pro Lys Arg Gly Lys Lys Gly - # Ala Val Ala Glu Asp Gly Asp    Glu    #   15    -      Leu Arg Thr Glu Pro Glu Ala Lys - # Lys Ser Lys Thr Ala Ala Lys    Lys    #                 30    -      Asn Asp Lys Glu Ala Ala Gly Glu - # Gly Pro Ala Leu Tyr Glu Asp    Pro    #             45    -      Pro Asp Gln Lys Thr Ser Pro Ser - # Ala Lys Pro Ala Thr Leu Lys    Ile    #         60    -      Cys Ser Trp Asn Val Asp Gly Leu - # Arg Ala Trp Ile Lys Lys Lys    Gly    #     80    -      Leu Asp Trp Val Lys Glu Glu Ala - # Pro Asp Ile Leu Cys Leu Gln    Glu    #   95    -      Thr Lys Cys Ser Glu Asn Lys Leu - # Pro Ala Glu Leu Gln Glu Leu    Pro    #                110    -      Gly Leu Ser His Gln Tyr Trp Ser - # Ala Pro Ser Asp Lys Glu Gly    Tyr    #            125    -      Ser Gly Val Gly Leu Leu Ser Arg - # Gln Cys Pro Leu Lys Val Ser    Tyr    #        140    -      Gly Ile Gly Asp Glu Glu His Asp - # Gln Glu Gly Arg Val Ile Val    Ala    #    160    -      Glu Phe Asp Ser Phe Val Leu Val - # Thr Ala Tyr Val Pro Asn Ala    Gly    #   175    -      Arg Gly Leu Val Arg Leu Glu Tyr - # Arg Gln Arg Trp Asp Glu Ala    Phe    #                190    -      Arg Lys Phe Leu Lys Gly Leu Ala - # Ser Arg Lys Pro Leu Val Leu    Cys    #            205    -      Gly Asp Leu Asn Val Ala His Glu - # Glu Ile Asp Leu Arg Asn Pro    Lys    #        220    -      Gly Asn Lys Lys Asn Ala Gly Phe - # Thr Pro Gln Glu Arg Gln Gly    Phe    #    240    -      Gly Glu Leu Leu Gln Ala Val Pro - # Leu Ala Asp Ser Phe Arg His    Leu    #   255    -      Tyr Pro Asn Thr Pro Tyr Ala Tyr - # Thr Phe Trp Thr Tyr Met Met    Asn    #                270    -      Ala Arg Ser Lys Asn Val Gly Trp - # Arg Leu Asp Tyr Phe Leu Leu    Ser    #            285    -      His Ser Leu Leu Pro Ala Leu Cys - # Asp Ser Lys Ile Arg Ser Lys    Ala    #        300    -      Leu Ala Ser Asp His Cys Pro Ile - # Thr Leu Tyr Leu Ala Leu    #    315    __________________________________________________________________________

What is claimed is:
 1. A method for identifying a premalignant ormalignant condition in a human subject comprising determining the levelof apurinic/apyrimidinic endonuclease (APE) in cells from a sample fromsaid human subject, wherein an elevated level of APE, as compared to theAPE level in corresponding normal cells, indicates a premalignant ormalignant condition in said human subject.
 2. The method of claim 1,wherein said sample is selected from the group consisting of skin,muscle, facia, brain, prostate, breast, endometrium, lung, pancreas,small intestine, blood cells, liver, testes, ovaries, cervix, colon,skin, stomach, esophagus, spleen, lymph node, bone marrow, kidney,peripheral blood, lymph fluid, ascites, serous fluid, pleural effusion,sputum, cerebrospinal fluid, lacrimal fluid, stool and urine.
 3. Themethod of claim 1, wherein said premalignancy or malignancy is in atissue selected from the group consisting of brain, lung, liver, spleen,kidney, lymph node, small intestine, pancreas, blood cells, colon,stomach, breast, endometrium, cervix, prostate, testicle, ovary, skin,head and neck, esophagus, bone marrow and blood tumor cells.
 4. Themethod of claim 1, wherein said deteimnig comprises evaluating APEprotein levels.
 5. The method of claim 1, wherein said determiningcomprises evaluating APE transcript levels.
 6. The method of claim 4,wherein said evaluating comprises an immunoassay.
 7. The method of claim5, wherein said evaluating comprises quantitative reversetranscription-polymerase chain reaction (RT-PCR).
 8. A method fordetermining the premalignant or malignant state of a cell comprisingdetermining the level of APE in said cell, wherein an elevated level ofAPE, as compared to the APE level in a corresponding normal cell,indicates a premalignant or malignant state in said cell.
 9. The methodof claim 8, wherein said determining comprises the steps of:(i)disrupting said cell; (ii) contacting said disrupted cell with anantibody that reacts immunologically with APE; and (iii) quantitatingthe amount of APE bound to said antibody.
 10. The method of claim 9,wherein said cell is disrupted by detergent lysis, freeze-thaw,sonication, osmotic shock or manual rupture.
 11. The method of claim 9,wherein said quantitating is by linked immunosorbant assay (ELISA). 12.The method of claim 9, wherein said quantitating is by radioimmunoassay(RIA).
 13. The method of claim 8, wherein said determining comprises thesteps of:(i) isolating mRNA from said cell; (ii) subjecting said mRNA toreverse transcription to produce cDNA; and (iii) quantitating APE cDNAby polymerase chain reaction (PCR).
 14. A method for diagnosis ofpremalignant or malignant condition in a human subject whichcomprises:(i) administering to the subject an imaging agent comprisingantibodies which react immunologically with APE bound to a label whichis detectable by an external scan of the subject; and (ii) externallyscanning the subject to determine whether there is a localizedconcentration of the imaging agent.
 15. The method of claim 14 whereinthe label is a radioactive label or is detectable by an X-ray, positronemission or magnetic resonance image scanning of the subject.